Purification of proteins using hydrophobic interaction chromatography

ABSTRACT

The present invention is directed to methods for purifying a protein of interest, e.g., an antibody, from a sample comprising the protein of interest and at least one impurity, e.g., an aggregate, by employing a hydrophobic interaction chromatography (HIC) method that allows for binding of both the protein of interest and the at least one impurity under strong binding conditions. The present invention is based, at least in part, on the finding that both flow through and bind-elute techniques can be combined to achieve greater purification and recovery of a protein of interest, e.g., an antibody, under isocratic wash conditions and strong binding conditions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/567,570, filed Dec. 11, 2014, which is a continuation of U.S. patentapplication Ser. No. 14/077,574, filed Nov. 12, 2013, now U.S. Pat. No.8,946,395, issued Feb. 3, 2015, which is related and claims priority toU.S. Provisional Application No. 61/893,131, filed Oct. 18, 2013, theentire contents of each of which are hereby incorporated herein byreference.

FIELD OF INVENTION

The instant invention relates to the field of protein production andpurification, and in particular to compositions and processes forreducing the levels of impurities, including process-related impurities(e.g., host cell proteins and media components) and/or product-relatedsubstances (e.g., product charge variants, aggregates, and fragments).

BACKGROUND OF THE INVENTION

Hydrophobic interaction chromatography (HIC) is a purification techniquethat exploits the interaction of HIC media with hydrophobic regionspresent on a protein of interest, such as an antibody, and/or thosepresent on an impurity to separate a protein of interest present in asample mixture. HIC is often utilized in either a bind-elute mode, inwhich the protein of interest remains bound to HIC media until elutedduring an elution phase, or a flow through mode, in which the protein ofinterest flows through the column while the impurity binds to the media.

Recently, a chromatographic method termed “weak partitioning mode” hasbeen described for the purification of proteins (U.S. Pat. No.8,067,182). According to U.S. Pat. No. 8,067,182, this method allows forthe binding of both product and impurity and is defined by aintermediate partition coefficient (Kp) for the product. Compared to theflow-through mode, in which the Kp for the product is typically low(e.g., <0.1), thereby allowing the product to flow through the columnwhile the impurity is bound, and the bind-elute mode, in which the Kpfor the product is typically high (e.g., >20), thereby allowing theproduct to remain bound until eluted during an elution phase, in theweak portioning mode, the Kp for the product is in the range of 0.1-20.

Importantly, U.S. Pat. No. 8,067,182 teaches the criticality of this Kprange. Specifically, U.S. Pat. No. 8,067,182 teaches that Kp valuesgreater than 20 result in a decreased load challenge at the point ofcontaminant breakthrough as the product begins to compete with thecontaminant for binding sites on the media. In addition, U.S. Pat. No.8,067,182 teaches that Kp values greater than 20 result in decreasedproduct recovery in that the isocratic wash conditions are not effectiveat washing the bound product off the column in a reasonable number ofwash volumes. Accordingly, U.S. Pat. No. 8,067,182, stresses thecriticality of a Kp range to achieve desired purification (see columns 9and 10).

When applied to HIC, the weak partitioning mode described in U.S. Pat.No. 8,067,182 requires an even more narrow Kp range. As set forth inExample 4, weak partitioning for HIC required a Kp less than 10.Patentees report that HIC performance deteriorates with respect to bothcontaminant reduction and product recovery at stronger bindingconditions.

SUMMARY OF THE INVENTION

The present invention is directed to methods for purifying a protein ofinterest, e.g., an antibody, from a sample including the protein ofinterest and at least one impurity, e.g., an aggregate, by employing anovel hydrophobic interaction chromatography (HIC) method. The presentinvention is based, at least in part, on the finding that both flowthrough and bind-elute techniques can be combined to achieve greaterpurification and recovery of a protein of interest. Moreover, thepresent invention is predicated, at least in part, on the surprisingfinding that such methodology can be employed under isocratic washconditions and at stronger binding conditions than previouslyappreciated, for example, at a Kp greater than 10, so as to achievegreater purification and recovery.

In one aspect, the present invention is directed to a method forproducing a preparation including a protein of interest and having areduced level of at least one impurity, said method comprising: (a)contacting a sample including the protein of interest and at least oneimpurity, to a hydrophobic interaction chromatography (HIC) media, inthe presence of a load buffer such that (i) a portion of the protein ofinterest binds to the HIC media and (ii) a substantial portion of the atleast one impurity binds to the HIC media; (b) collecting a flow throughfraction including the protein of interest unbound to the HIC media; (c)washing the HIC media with a wash buffer that is substantially the sameas the load buffer such that a substantial portion of the protein ofinterest bound to the HIC media is released from the media; and (d)collecting a wash fraction including the protein of interest releasedfrom the HIC media, wherein each of the flow through and wash fractionsinclude the protein of interest and have a reduced level of the at leastone impurity.

In various embodiments, the portion of the protein of interest binds tothe HIC media at a Kp of greater than 10, 15, 20, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250 or 300.For example, in various embodiments, the portion of the protein ofinterest binds to the HIC media at a Kp of greater than 10, the portionof the protein of interest binds to the HIC media at a Kp of greaterthan 20, or the portion of the protein of interest binds to the HICmedia at a Kp of greater than 100.

In a particular embodiment, the protein of interest is adalimumab.

In one embodiment, a substantial portion of the impurity bound to theHIC media remains bound upon washing with the wash buffer. In oneembodiment, the flow through and/or wash fractions are substantiallyfree of the at least one impurity.

In one embodiment, the at least one impurity is an aggregate of theprotein of interest, for example, selected from the group consisting ofa multimer, a dimer, a trimer, a tetramer, an oligomer and other highmolecular weight species. In a particular embodiment, the protein ofinterest is adalimumab and the at least one impurity is an aggregate ofadalimumab. For example, the aggregate may be selected from the groupconsisting of multimer 1, multimer 2 and multimer 3.

In another embodiment, the impurity is a process-related impurity or aproduct-related substance. For example, the impurity may be aprocess-related impurity selected from the group consisting of a hostcell protein, a host cell nucleic acid, a media component, and achromatographic material. Alternatively, the impurity may be aproduct-related substance selected from the group consisting of a chargevariant, an aggregate of the protein of interest, a fragment of theprotein of interest and a modified protein.

In a particular embodiment the impurity is an acidic or basic variant,for example, of adalimumab. In a particular embodiment, the basicvariant is a lysine variant species, for example, an antibody, orantigen-binding portion thereof, having heavy chains with either zero,one or two C-terminal lysines. In another embodiment, the impurity is anacidic species (AR), for example, selected from the group consisting ofa charge variant, a structure variant, a fragmentation variant, aprocess-related impurity and a product-related impurity. In a particularembodiment, the acidic species is AR1 and the charge variant is adeamidation variant, a glycation variant, an afucosylation variant, aMGO variant and/or a citric acid variant. In another embodiment, theacidic species is AR1 and the structure variant is a glycosylationvariant and/or an acetonation variant. In yet another embodiment, theacidic species is AR1 and the fragmentation variant is a Fab fragmentvariant, a C-terminal truncation variant or a variant missing a heavychain variable domain. In yet a further embodiment, the acidic speciesis AR2 and the charge variant comprises a deamidation variant and/orglycation variant.

In a particular embodiment, the impurity is a fragment such as an Fc ora Fab fragment. In another embodiment, the impurity is a modifiedprotein such as a deamidated protein or glycosylated protein.

In one embodiment, the protein of interest is an antibody orantigen-binding fragment thereof, a soluble protein, a membrane protein,a structural protein, a ribosomal protein, an enzyme, a zymogen, anantibody molecule, a cell surface receptor protein, a transcriptionregulatory protein, a translation regulatory protein, a chromatinprotein, a hormone, a cell cycle regulatory protein, a G protein, aneuroactive peptide, an immunoregulatory protein, a blood componentprotein, an ion gate protein, a heat shock protein, an antibioticresistance protein, a functional fragment of any of the precedingproteins, an epitope-containing fragment of any of the precedingproteins, and combinations thereof.

In a particular embodiment, the protein of interest is an antibody orantigen-binding fragment thereof such as a humanized antibody orantigen-binding portion thereof, a human antibody or antigen-bindingportion thereof, a chimeric antibody or antigen-binding portion thereof,or a multivalent antibody. In one embodiment, the antibody, orantigen-binding fragment thereof, comprises a heavy chain constantregion selected from the group consisting of IgG1, IgG2, IgG3, IgG4,IgM, IgA and IgE constant regions. In another embodiment, the antibody,or antigen-binding fragment thereof is selected from the groupconsisting of a Fab fragment, a F(ab′)2 fragment, a single chain Fvfragment, an SMIP, an affibody, an avimer, a nanobody, and a singledomain antibody.

In one embodiment, the methods of the invention further includerepeating steps (a)-(d) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 or 20 times using the flow through fraction, wash fraction,or combination thereof having a reduced level of the at least oneimpurity. In certain embodiments, the flow through fraction and the washfraction are combined.

In one embodiment, the portion of the protein of interest that binds tothe HIC media is at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80% or at least about 90% of the protein of interest in thesample. Alternatively or in combination, the substantial portion of theprotein of interest released from the HIC media upon washing with thewash buffer is about at least 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90% or about 100% of the amount ofprotein of interest bound to the HIC media. Alternatively or incombination, the substantial portion of the at least one impurity thatbinds to the HIC media is at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95% or about 100% of the at least one impurity in thesample.

In certain embodiments, the accumulative yield of the protein ofinterest in the flow through fraction and/or wash fraction is at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%. Alternatively orin combination, the accumulative yield of the protein of interest in anyone flow through fraction and/or wash fraction is at least about 4%, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85, at least about 90%, at least about 95% or about100%. Alternatively or in combination, the reduced level of the at leastone impurity of the flow through fraction and/or wash fraction is atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95% or about 100% ofthe at least one impurity in the sample.

In certain embodiments, the accumulative aggregate reduction of the atleast one impurity in any one flow through fraction and/or wash fractionis at least about 0.1%, at least about 0.2%, at least about 0.5%, atleast about 1.0%, at least about 2.0%, at least about 3.0%, at leastabout 4.0%, at least about 5.0%, at least about 10.0%, or at least about20.0%. Alternatively or in combination, the accumulative aggregatereduction of the at least one impurity in the flow through fractionand/or wash fraction is at least about 0.1%, at least about 0.2%, atleast about 0.5%, at least about 1.0%, at least about 2.0%, at leastabout 3.0%, at least about 4.0%, at least about 5.0%, at least about10.0%, or at least about 20.0%.

In certain embodiments, the at least one impurity binds to the HIC mediaat a Kp of greater than 250, greater than 300, greater than 400, greaterthan 500, greater than 600, greater than 700, greater than 800, greaterthan 900, or greater than 1000. In certain embodiments, the protein ofinterest and the at least one impurity have a Kp ratio less than 1:10,1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 or 1:2.

In certain embodiments, the K_(d) for the binding of the protein ofinterest to the HIC media is at least about 0.2, at least about 0.3, atleast about 0.4, at least about 0.5, or at least about 0.6.Alternatively or in combination, the K_(d) for the binding of the atleast one impurity to the HIC media is less than or equal to about0.001, about 0.005, about 0.01, about 0.02, about 0.05, about 0.1, about0.15 or about 0.2. In particular embodiments, the K_(d) for the bindingof the protein of interest to the HIC media is less than 50, 45, 40, 35,30, 25, 20, 15, 10 or 5 times the K_(d) for the binding of the at leastone impurity to the HIC media.

In certain embodiments, the protein of interest has a Qmax of at leastabout 20, at least about 30, at least about 40, at least about 50, atleast about 60 or at least about 100. In certain embodiments, the atleast one impurity has a Qmax of at least about 2, at least about 5, atleast about 10, at least about 20, at least about 30 or at least about40.

In certain embodiments, the HIC media comprises at least one hydrophobicligand. For example, the HIC media may be selected from the groupconsisting of alkyl-, aryl-ligands, and combinations thereof. Forexample, the HIC media may be selected from the group consisting ofbutyl, hexyl, phenyl, octyl, or polypropylene glycol ligands. In aparticular embodiment, the HIC media is selected from the groupconsisting of CaptoPhenyl, Phenyl Sepharose™ 6 Fast Flow with low orhigh substitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™High Performance, Fractogel™ EMD Propyl, Fractogel™ EMD Phenyl,Macro-Prep™ Methyl, Macro-Prep™ t-Butyl, WP HII-Propyl (C3)™, Toyopearl™ether, Toyopearl™ phenyl, Toyopearl™ butyl, ToyoScreen PPG, ToyoScreenPhenyl, ToyoScreen Butyl, ToyoScreen Hexyl, HiScreen Butyl FF, HiScreenOctyl FF, and Tosoh Hexyl. In one embodiment, the HIC media is a column.

In various embodiments, the load buffer and/or wash buffer comprise asalt selected from the group consisting of ammonium sulfate, sodiumsulfate, sodium chloride, ammonium chloride, sodium bromide or acombination thereof. In a particular embodiment, the load buffer and thewash buffer include a sulfate salt, a citrate salt, or a combinationthereof. For example, the sulfate salt may be ammonium sulfate or sodiumsulfate. In certain embodiments, the citrate salt is sodium citrate. Invarious embodiments, the load buffer and/or the wash buffer comprise acation selected from the group consisting of Ba²⁺, Ca²⁺, Mg²⁺, Li⁺, Cs⁺,Na⁺, K⁺, Rb⁺, and NH₄ ⁺, and/or an anion selected from the groupconsisting of PO₄ ³⁻, SO₄ ²⁻, CH₃CO₃ ⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, andSCN⁻ or a combination thereof.

In one embodiment, the salt has a concentration of between about 50 mMand 2000 mM. In a particular embodiment, the load buffer and the washbuffer have a pH between about 4.0 and 8.5 or between about 5.0 and 7.0.In certain embodiments, the load buffer and the wash buffer have a pH ofabout 4.0, about 4.5, about 5.0, about 5.5, about 6, about 6.5, about7.0, about 7.5, about 8.0, or about 8.5.

In one embodiment, the load buffer and the wash buffer are the same. Inone embodiment, the load buffer and the wash buffer are substantiallythe same. For example, the salt concentration and/or the pH of the washbuffer may be within about 20%, 15%, 10% or 5% of the saltconcentration, and/or pH of the loading buffer.

In certain embodiments, about 100 g to about 800 g of the sample arecontacted per one liter of HIC media. Alternatively or in combination,about 0.2 g to about 120 g of the at least one impurity is contacted perone liter of HIC media. In certain embodiments, the sample has a proteinconcentration of about 2 mg/ml to about 50 mg/ml. In certainembodiments, the sample has a protein of interest concentration of about2 mg/ml to about 50 mg/ml. Alternatively or in combination, theconcentration of the at least one impurity in the sample is about 0.01to about 5.0 mg/ml.

In various embodiments, the level of the at least one impurity isreduced by at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% of the at least one impurity in the sample.

In one embodiment, the at least one impurity is a host cell protein. Forexample, the host cell protein may be reduced by at least 0.25, at least0.5, at least 0.75, at least 1.0, at least 1.25 or at least 1.5 logreduction fraction.

In one embodiment, the HIC media has a dynamic binding capacity of atleast about 2 g, at least about 5 g, at least about 10 g, at least about20 g, at least about 30 g, at least about 40 g, at least about 50 g, atleast about 60 g, at least about 70 g, at least about 90 g, or at leastabout 100 g of sample per one liter of media.

In one embodiment, a precursor sample including the protein of interesthas been subjected to affinity chromatography to generate the sample.Alternatively or in combination, the preparation including a protein ofinterest and having a reduced level of one impurity is subjected toaffinity chromatography. In such embodiments, affinity chromatographymay be performed using affinity chromatographic media selected from thegroup consisting of Protein A, G, A/G, L media, and MabSuRe Protein Amedia.

In one embodiment, a precursor sample including the protein of interesthas been subjected to ion exchange chromatography to generate thesample. Alternatively or in combination, the preparation including aprotein of interest and having a reduced level of one impurity issubjected to ion exchange chromatography. In such embodiments, ionexchange chromatography may be performed using ion exchangechromatography media selected from the group consisting of (i) a cationexchange media, for example, comprising carboxymethyl (CM),sulfoethyl(SE), sulfopropyl(SP), phosphate(P) or sulfonate(S) ligands,and (ii) an anion exchange media, for example, comprisingdiethylaminoethyl (DEAE), quaternary aminoethyl (QAE) or quaternaryamine (Q) group ligands.

In one embodiment, a precursor sample including the protein of interesthas been subjected to mixed mode chromatography to generate the sample.Alternatively or in combination, the method involves subjecting thepreparation including a protein of interest and having a reduced levelof one impurity to mixed mode chromatography, for example, usingCaptoAdhere resin.

In one embodiment, a precursor sample including the protein of interesthas been subjected to a filtration step to generate the sample.Alternatively or in combination, the method involves subjecting thepreparation including a protein of interest and having a reduced levelof one impurity to a filtration step, for example, a depth filtrationstep, a nanofiltration step, an ultrafiltration step, and an absolutefiltration step, or a combination thereof.

In one aspect, the present invention is directed to a pharmaceuticalcomposition including the preparation produced by any of the foregoingmethods.

In another aspect, the present invention is directed to a method forproducing a preparation including adalimumab and having a reduced levelof at least one aggregate, by (a) contacting a sample of adalimumab andat least one aggregate, to a HIC media, in the presence of a load buffersuch that (i) a portion of the adalimumab in the sample binds to the HICmedia and (ii) a substantial portion of the at least one aggregate bindsto the HIC media; (b) collecting a flow through fraction of theadalimumab unbound to the HIC media; (c) washing the HIC media with awash buffer that is substantially the same as the load buffer such thata substantial portion of the adalimumab bound to the HIC media isreleased from the media; and (d) collecting a wash fraction of theadalimumab released from the HIC media, wherein each of the flow throughand wash fractions comprise adalimumab and have a reduced level of theat least one aggregate.

In one embodiment of the foregoing method, adalimumab binds to the HICmedia at a Kp of greater than 10, 15, 20, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250 or 300. Forexample, adalimumab binds to the HIC media at a Kp of greater than 10.Alternatively, adalimumab binds to the HIC media at a Kp of greater than20. Alternatively, adalimumab binds to the HIC media at a Kp of greaterthan 90.

In a particular embodiment, the aggregate is multimer 1, multimer 2 ormultimer 3.

In various embodiments, the sample includes between 200 g and 700 gprotein per liter of HIC media. In certain embodiments, the HIC media isselected from the group consisting of GE CaptoPhenyl, Tosoh Hexyl, GEButyl FF, Butyl, Hexyl, Phenyl, Octyl, GE Butyl FF, PPG. In certainembodiments, the load buffer and the wash buffer include ammoniumsulfate, sodium sulfate, sodium citrate, or a combination thereof.Alternatively or in combination, the pH of the load buffer and the washbuffer is between 5 and 7. Alternatively or in combination, the saltconcentration of the load buffer and the wash buffer is between about150 mM and 1000 mM.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a low-aggregate composition and a pharmaceuticalacceptable carrier.

In one aspect, the present invention provides a pharmaceuticalcomposition comprising a preparation of adalimumab produced by theforegoing methods and a pharmaceutically acceptable carrier. In anotheraspect, the present invention provides a pharmaceutical compositioncomprising a low-aggregate composition of adalimumab and apharmaceutically acceptable carrier. For example, the composition mayinclude less than 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%,1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1% of aggregates, e.g., MM1, MM2 andMM3. Alternatively, the composition may include less than 2%, 1.5%,1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1% of aggregates, e.g., MM1, MM2 andMM3. Alternatively, the composition may include less than 1%, 0.5%, 0.1%of aggregates, e.g., MM1, MM2 and MM3.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 depicts a size exclusion chromatography (SEC) chromatogram usedto determine the molecular weight distribution of a sample ofadalimumab. In combination with multi-angle light scattering (MALS)analysis (data not shown), the apparent molecular weight of each peakwas determined and identified as a multimer or the reference standard asindicated. Multimer 1 (MM1), Multimer 2 (MM2) and Multimer 3 (MM3) wereidentified as depicted.

FIGS. 2A-2B depicts schematic chromatograms for two modes ofchromatographic operation: bind-elute mode (FIG. 2A) and flow-throughmode (FIG. 2B). In the bind-elute mode, there is strong binding of theprotein of interest and the impurity. Elution conditions are chosen toselectively elutes the protein of interest. In the flow-through modethere is weak binding of the product and strong binding of the impurity.

FIG. 3 depicts selection of operating conditions appropriate for anantibody:media:buffer combination. A sample was loaded at 20 g/L and alinear gradient elution was performed over 20 CVs to identify the saltconcentration at the monomer and aggregate peak. The salt concentrationat or near the elution peak of the monomer is the concentration at whichthe monomer is eluted from the HIC media.

FIG. 4 depicts a process chromatogram for the HIC purification ofAdalimumab, wherein a GE CaptoPhenyl column was equilibrated at 1.1 MAmSO₄ pH 7.0 (Tris/Acetate) for 10 CVs, Adalimumab was prepared at 1.1 MAmSO₄ and loaded to the column at 20 g-protein/L of media. The columnwas then washed with 10 CVs of the equilibration buffer and a lineargradient from 1.1 M to 0 M AmSO₄ pH 7.0 (Tris/Acetate) over 20 CVs wasperformed. See Example 1.

FIG. 5 depicts a process chromatogram for the HIC purification ofAdalimumab, wherein a GE CaptoPhenyl column was equilibrated with 400 mMNaCit pH 5.6 for 10 CVs, Adalimumab was prepared at 400 mM NaCit pH 5.6and then loaded to the column at 500 g-protein/L-media. Finally, thecolumn was washed with 7 CVs of the equilibration buffer. See Example 1.

FIG. 6 depicts results of an experiment wherein a feed stream wasserially diluted to cover a range of load concentrations from 4 to 15mg/mL and loaded at 500 g/L to a CaptoPhenyl column in 400 mM NaCit pH5.6. The results indicate the impact that the concentration of loadedprotein can have on aggregate reduction. See Example 7.

FIG. 7 depicts the effect of aggregate load concentration on dynamicbinding capacity and aggregate clearance. The column is conditioned andloaded at different sample load concentrations. The flow-through isfractionated to determine the product quality at different times duringthe load and breakthrough. Using protein mass and product quality foreach of the collected fractions, the accumulative impurity (e.g.,aggregate) can be calculated. The accumulative impurity of thepreparation is reduced when the concentration of the aggregate in theload is reduced, even when the total load is unchanged (e.g., 500 g/L).See Example 13.

FIGS. 8A-8C depicts the effect of overall load protein concentration inthe sample. The column is conditioned and loaded at different sampleload concentrations. The flow through is fractionated to determine theproduct quality at different times during the load and breakthrough(FIG. 8A). Using protein mass and product quality for each of thecollected factions, the accumulative aggregate impurity can becalculated. The accumulative aggregate impurity of the preparation isreduced when the protein concentration of the sample is reduced. TheEquilibrium Binding Isotherms for both the monomer and aggregate showthat for all of the loading conditions (FIG. 8B and FIG. 8C), themonomer was in the non-linear part of its binding isotherm (e.g.,equilibrium binding capacity is independent of monomer concentration),and the aggregate was in or near the linear part of its binding isotherm(e.g., equilibrium binding capacity is dependent on aggregateconcentration). Aggregate dynamic binding capacity=f(C_(o), t). SeeExample 13.

FIG. 9 depicts the modulation of the recovery-yield for a given targetimpurity clearance by diluting the load material to a specific range.See Example 13.

FIG. 10 depicts dynamic binding capacity (DBC), conventionally measuredat 10% breakthrough, as greater than the equilibrium binding capacity(EBC), based on the data presented in FIG. 5. See Example 1.

FIG. 11 depicts a dynamic binding capacity of >75 g/L. Following anisocratic wash and regeneration step, the remaining protein bound to theresin is <35 g/L.

FIGS. 12A-12B depicts determination of Apparent Binding Capacity (FIG.12A), and Actual Binding Capacity (FIG. 12B). See Example 12.

FIGS. 13A-13B depict the results of experiments wherein aliquots ofresin are incubated with a load covering a range of proteinconcentrations at room temperature for 3 hours, after which the proteinsolution is then removed, and replaced with equilibration buffer (Washsimulation) and incubated at room temperature for 3 hours (repeated,Wash II). After each incubation, the concentration of the proteinsolution is measured and used to calculated the amount of protein ((FIG.13A) monomer D2E7, (i.e., Adalimumab), and (FIG. 13B) aggregate D2E7)bound to the resin (g protein/L resin) and plotted against theconcentration of the protein solution at the end of the incubation(e.g., equilibrium). See Example 11.

FIGS. 14A-14B depict the results outlined in FIGS. 13A-13B, highlightingthe fact that at initial equilibrium a significant amount ofmonomer/aggregate is bound to the resin. However, after the proteinsolution is replaced with equilibration buffer (see arrow), the monomerdesorbs from the resin and back into solution, whereas the aggregateremains bound. See Example 11.

FIGS. 15A-15B depict a determination of the binding monomer andaggregate D2E7 (based on data provided in FIGS. 13A-13B) by fitting theexperimental equilibrium binding data to the Langmuir Isotherm using theequation: q=(q_(max)× C_(equil))/(K_(a)+C_(equil)); where q=amount ofprotein bound to resin [=] g/L-resin; q_(max)=maximum amount of proteinbound to resin [=] g/L-resin; C_(equil)=solution concentration ofprotein [=] g/L-soln; and K_(a)=equilibrium dissociation constant. SeeExample 11.

FIG. 16 depicts the comparison of Apparent and Actual bound proteinunder flow conditions (partial partitioning) as a function of saltconcentration. Binding of the protein of interest is significant >10g/L. The majority (>65%) of this monomer bound during the load desorbsduring the isocratic wash (i.e., reversibly bound). The mass balance ofthe impurity demonstrates irreversible binding. See Example 12.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for purifying a protein ofinterest, e.g., an antibody, from a sample comprising the protein ofinterest and at least one impurity, e.g., an aggregate, by employing anovel hydrophobic interaction chromatography (HIC) method. The presentinvention is based, at least in part, on the finding that both flowthrough and bind-elute techniques can be combined to achieve greaterpurification and recovery of a protein of interest, e.g., an antibody.Moreover, the present invention is predicated, at least in part, on thesurprising finding that such methodology can be employed under isocraticwash conditions and at stronger binding conditions than previouslyappreciated, for example, at a Kp greater than 10 or at a Kp greaterthan 20, so as to achieve greater purification and recovery.

In one aspect, the present invention provides a method for producing apreparation including a protein of interest, e.g., an antibody such asadalimumab, and having a reduced level of at least one impurity, e.g.,an aggregate, by (a) contacting a sample including the protein ofinterest and at least one impurity, to a hydrophobic interactionchromatography media, in the presence of a load buffer such that (i) aportion of the protein of interest binds to the hydrophobic interactionchromatography (HIC) media and (ii) a substantial portion of the atleast one impurity binds to the HIC media; (b) collecting a flow throughfraction including the protein of interest unbound to the HIC media; (c)washing the HIC media with a wash buffer that is substantially the sameas the load buffer such that a substantial portion of the protein ofinterest bound to the HIC media is released from the media; and (d)collecting a wash fraction including the protein of interest releasedfrom the HIC media, wherein each of the flow through and wash fractionsinclude the protein of interest and have a reduced level of the at leastone impurity. In a particular embodiment, the portion of the protein ofinterest binds to the HIC media at a Kp of greater than 10. In anotherembodiment, the portion of the protein of interest binds to the HICmedia at a Kp of greater than 20. In another embodiment, the portion ofthe protein of interest binds to the HIC media at a Kp of greater than100.

In certain embodiments, the purification strategies of the presentinvention may include one or more chromatography and/or filtration stepsto achieve a desired degree of purification prior to exposure of thesample comprising the protein of interest, e.g., an antibody such asadalimumab, to the HIC media. For example, in certain embodiments, suchpre-HIC chromatography step(s) can include one or more steps ofchromatography and/or filtration. In one embodiment the chromatographyis ion exchange chromatography, mixed mode chromatography, and/oraffinity chromatography. In another embodiment, the filtration step isdepth filtration, nanofiltration, ultrafiltration and/or absolutefiltration. In certain embodiments, the purification strategies of thepresent invention may include one or more additional chromatographyand/or filtration steps after the HIC purification step. For example, incertain embodiments, such post-HIC chromatography step(s) can includeone or more steps of chromatography and/or filtration. In oneembodiment, the chromatography is ion exchange chromatography, mixedmode chromatography, and/or affinity chromatography. In anotherembodiment, the filtration step is depth filtration, nanofiltration,ultrafiltration and/or absolute filtration.

In addition, in certain embodiments, the present invention is directedtoward pharmaceutical compositions comprising one or more proteins ofinterest purified by methods described herein. In a particularembodiment, the present invention is directed to a pharmaceuticalcomposition comprising adalimumab and having a reduced level ofaggregates.

Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms, for example, those characterized by “a” or“an”, shall include pluralities, e.g., one or more impurities. In thisapplication, the use of “or” means “and/or”, unless stated otherwise.Furthermore, the use of the term “including,” as well as other forms ofthe term, such as “includes” and “included”, is not limiting. Also,terms such as “element” or “component” encompass both elements andcomponents comprising one unit and elements and components that comprisemore than one unit unless specifically stated otherwise.

As used herein, the term “sample”, refers to a liquid compositionincluding the protein of interest and one or more impurities. In aparticular embodiment, the sample is a “clarified harvest”, referring toa liquid material containing a protein of interest, for example, anantibody of interest such as adalimumab, that has been extracted fromcell culture, for example, a fermentation bioreactor, after undergoingcentrifugation to remove large solid particles and subsequent filtrationto remove finer solid particles and impurities from the material.

In various embodiments, the sample may be partially purified. Forexample, the sample may have already been subjected to any of a varietyof art recognized purification techniques, such as chromatography, e.g.,ion exchange chromatography, mixed mode chromatography, and/or affinitychromatography, or filtration, e.g., depth filtration, nanofiltration,ultrafiltration and/or absolute filtration.

The term “precursor sample”, as used herein refers to a liquidcomposition containing the protein of interest and, optionally, one ormore impurities, either derived from the clarified harvest, or apartially purified intermediate sample that is subject to a purificationor treatment step prior to being subjected to HIC. Impurities in aprecursor sample may be derived from the production, purification ortreatment of the protein of interest prior to subjecting the resultingsample to HIC.

The term “protein of interest”, as used herein refers to a targetprotein present in a sample, purification of which is desired. Invarious embodiment, the protein of interest is an antibody orantigen-binding fragment thereof, a soluble protein, a membrane protein,a structural protein, a ribosomal protein, an enzyme, a zymogen, anantibody molecule, a cell surface receptor protein, a transcriptionregulatory protein, a translation regulatory protein, a chromatinprotein, a hormone, a cell cycle regulatory protein, a G protein, aneuroactive peptide, an immunoregulatory protein, a blood componentprotein, an ion gate protein, a heat shock protein, an antibioticresistance protein, a functional fragment of any of the precedingproteins, an epitope-containing fragment of any of the precedingproteins, and combinations thereof. In a particular embodiment, theprotein of interest is a monomer.

In a particular embodiment, the protein of interest is an antibody, oran antigen binding portion thereof. The term “antibody” includes animmunoglobulin molecule comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region(CH). The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as LCVR or VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH and VL regions can be further subdivided into regionsof hypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term “antibody”, as usedherein, also includes alternative antibody and antibody-like structures,such as, but not limited to, dual variable domain antibodies (DVD-Ig).

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., hIL-12, hTNFα, or hIL-18). It hasbeen shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment comprisingthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment comprising the VH and CH1 domains;(iv) a Fv fragment comprising the VL and VH domains of a single arm ofan antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546,the entire teaching of which is incorporated herein by reference), whichcomprises a VH domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883, the entire teachings of which areincorporated herein by reference). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123, theentire teachings of which are incorporated herein by reference). Stillfurther, an antibody may be part of a larger immunoadhesion molecule,formed by covalent or non-covalent association of the antibody with oneor more other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101, the entire teaching of which isincorporated herein by reference) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058, the entire teaching of which is incorporatedherein by reference). Antibody portions, such as Fab and F(ab′)2fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein. In one aspect, the antigen binding portions arecomplete domains or pairs of complete domains.

The term “human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat, et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), e.g., in the CDRs and in particular CDR3. Themutations can be introduced using the “selective mutagenesis approach.”The human antibody can have at least one position replaced with an aminoacid residue, e.g., an activity enhancing amino acid residue which isnot encoded by the human germline immunoglobulin sequence. The humanantibody can have up to twenty positions replaced with amino acidresidues which are not part of the human germline immunoglobulinsequence. In other embodiments, up to ten, up to five, up to three or upto two positions are replaced. In one embodiment, these replacements arewithin the CDR regions. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes (see,e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295, theentire teaching of which is incorporated herein by reference) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.In certain embodiments, however, such recombinant antibodies are theresult of selective mutagenesis approach or back-mutation or both.

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds hTNFα is substantially free ofantibodies that specifically bind antigens other than hTNFα). Anisolated antibody that specifically binds hTNFα may bind TNFα moleculesfrom other species. Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals. A suitable anti-TNFαantibody is Adalimumab (AbbVie).

As used herein, the term “adalimumab,” also known by its trade nameHUMIRA® (AbbVie) refers to a human IgG₁ antibody that binds human tumornecrosis factor α (TNFα). In general, the heavy chain constant domain 2(CH2) of the adalimumab IgG-Fc region is glycosylated through covalentattachment of oligosaccharide at asparagine 297 (Asn-297). The lightchain variable region of adalimumab is provided herein as SEQ ID NO:1,and the heavy chain variable region of adalimumab is provided herein asSEQ ID NO:2. Adalimumab comprises a light chain variable regioncomprising a CDR1 of SEQ ID NO:7, a CDR2 of SEQ ID NO:5, and a CDR3 ofSEQ ID NO:3. Adalimumab comprises a heavy chain variable regioncomprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID NO:6 and CDR3 of SEQID NO:4. The nucleic acid sequence of the light chain variable region isset forth in SEQ ID NO:9. The nucleic acid sequence of the heavy chainvariable region is set forth in SEQ ID NO:10. The full length amino acidsequence of the light chain is set forth as SEQ ID NO:11 and the fulllength amino acid sequence of the heavy chain is set forth as SEQ IDNO:12. Adalimumab is described in U.S. Pat. Nos. 6,090,382; 6,258,562;6,509,015; 7,223,394; 7,541,031; 7,588,761; 7,863,426; 7,919,264;8,197,813; 8,206,714; 8,216,583; 8,420,081; 8,092,998; 8,093,045;8,187,836; 8,372,400; 8,034,906; 8,436,149; 8,231,876; 8,414,894;8,372,401, the entire contents of each which are expressly incorporatedherein by reference in their entireties. Adalimumab is also described inthe “Highlights of Prescribing Information” for HUMIRA® (adalimumab)Injection (Revised January 2008) the contents of which are herebyincorporated herein by reference.

In one embodiment, adalimumab dissociates from human TNFα with a Kd of1×10⁻⁸ M or less and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance, and neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10⁻⁷ Mor less. In another embodiment, adalimumab dissociates from human TNFαwith a K_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, with aK_(off) of 1×10⁻⁴ s⁻¹ or less. In still another embodiment, adalimumabneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC50 of 1×10⁻⁸ M or less, an IC50 of 1×10⁻⁹ M or less or an IC50of 1×10⁻¹° M or less. The term “Koff”, as used herein, is intended torefer to the off rate constant for dissociation of an antibody from theantibody/antigen complex.

The term “impurity”, as used herein refers to any foreign orobjectionable molecule, including a biological macromolecule such as aDNA, an RNA, or a protein other than the protein of interest beingpurified. Exemplary impurities include, for example, protein variants,such as aggregates, high molecular weight species, low molecular weightspecies and fragments, and deamidated species; host cell proteins;proteins that are part of an absorbent used for affinity chromatography(e.g. Protein A); endotoxins; and viruses.

The methods of the invention serve to generate a preparation comprisinga protein of interest and having a reduced level of impurity. As usedherein a “reduced level of impurity” refers to a composition comprisingreduced levels of an impurity as compared to the levels of the impurityin the sample prior to purification by the methods of the presentinvention. In another embodiment, the methods of the invention generatea preparation comprising a protein of interest and having a reducedlevel of total impurity. As used herein a “reduced level of totalimpurity” refers to a composition comprising reduced levels of totalimpurity as compared to the levels of the impurity in the sample priorto purification by the methods of the present invention. In oneembodiment, a preparation having a reduced level of total impurity isfree of impurities or substantially free of impurities.

The present invention is further directed to low impurity compositionsand methods of generating the same, for example, low impuritycompositions of adalimumab. The term “low impurity composition,” as usedherein, refers to a composition comprising a protein of interest,wherein the composition contains less than about 15% total impurities.For example, a low impurity composition may contain about 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, or lesstotal impurities. In a particular embodiment, a low impurity compositioncomprises about 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%,1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%, or less total impurities.

The term “non-low impurity composition,” as used herein, refers to acomposition comprising a protein of interest, which contains more thanabout 15% total impurity. For example, a non-low impurity compositionmay contain about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,or more total impurities.

In one embodiment, a low impurity composition has improved biologicaland functional properties, including increased efficacy in the treatmentor prevention of a disorder in a subject, e.g., a disorder in which TNFαactivity is detrimental, as compared to a non-low impurity composition.In one embodiment, the low impurity composition comprises an anti-TNFαantibody, or antigen-binding portion thereof, such as adalimumab or afragment thereof. For example, in one embodiment, a low impuritycomposition comprising an antibody, or antigen-binding portion thereof,exhibits increased cartilage penetration, decreased bone erosion, and/orreduced cartilage destruction, as compared to a non-low impuritycomposition comprising the same antibody or antigen binding portionthereof, when administered to a subject suffering from a disorder inwhich TNFα activity is detrimental.

As used herein, the term “increased cartilage penetration” refers toincreased penetration of cartilage in vivo by a low impurity compositionas compared to a non-low impurity composition comprising the sameantibody or antigen binding portion thereof.

As used herein, the term “reduced cartilage destruction” refers tomeasurable decrease in destruction of cartilage tissue in vivo by a lowimpurity composition as compared to a non-low impurity compositioncomprising the same antibody or antigen binding portion thereof. As usedherein, the term “decreased bone erosion” refers to measurable decrease,in vivo, of the erosion of bone tissue by a low impurity composition ascompared to a non-low aggregate composition comprising the same antibodyor antigen binding portion thereof. For example, an in vivo model of adisease or disorder in which TNFα activity is detrimental, e.g., a mousemodel of arthritis, can be used to measure cartilage penetration, boneerosion, and/or cartilage destruction by a composition comprising ananti-TNFα antibody or antigen binding portion thereof. One non-limitingexample of an art-recognized mouse model of arthritis is the human TNFtransgenic 197 mouse model of arthritis (TNF-Tg197) (see Keffer, J. etal., EMBO J (1991) 10:4025-4031 for further description of the TNF-Tg197model of arthritis).

In another embodiment, a low impurity composition comprising anantibody, or antigen-binding portion thereof, exhibits increasedprotection against the development of arthritic scores and/orhistopathology scores as compared to a non-low impurity composition whenadministered to an animal model of arthritis, e.g., the TNF-Tg197 modelof arthritis. As used herein, “arthritic scores” refer to signs andsymptoms of arthritis in an animal model of arthritis. As used herein,“histopathology scores” refer to radiologic damage involving cartilageand bone as well as local inflammation.

In another embodiment, a low impurity composition comprising anantibody, or antigen-binding portion thereof, exhibits reduced synovialproliferation, reduced cell infiltration, reduced chondrocyte death,and/or reduced proteoglycan loss as compared to a non-low impuritycomposition. In another embodiment, a low impurity compositioncomprising an anti-TNFα antibody, or antigen-binding portion thereof,exhibits increased TNFα affinity as compared to a non-low impuritycomposition.

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which TNFαactivity is detrimental is a disorder in which inhibition of TNFαactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, or synovial fluid of the subject), which can bedetected, for example, using an anti-TNFα antibody as described above.There are numerous examples of disorders in which TNFα activity isdetrimental. In one embodiment, the disorder in which TNFα activity isdetrimental is an autoimmune disorder. In one embodiment, the autoimmunedisorder is selected from the group consisting of rheumatoid arthritis,juvenile idiopathic arthritis, rheumatoid spondylitis, ankylosingspondylitis, psoriasis, osteoarthritis, gouty arthritis, an allergy,multiple sclerosis, psoriatic arthritis, autoimmune diabetes, autoimmuneuveitis, nephrotic syndrome, juvenile rheumatoid arthritis, Crohn'sdisease, ulcerative colitis, active axial spondyloarthritis (activeaxSpA) and non-radiographic axial spondyloarthritis (nr-axSpA).Disorders in which TNFα activity is detrimental are set forth in U.S.Pat. No. 6,090,382 and also in the Humira® Prescribing Information, thecontents of each of which are hereby incorporated herein by reference.The use of TNFα antibodies and antibody portions obtained using methodsof the invention for the treatment of specific disorders is discussed infurther detail below.

In a particular embodiment, the impurity is a process-related impurity.As used herein, the term “process-related impurity,” refers toimpurities that are present in a composition comprising a protein ofinterest but are not derived from the protein itself. Process-relatedimpurities include, but are not limited to, host cell proteins (HCPs),host cell nucleic acids, chromatographic materials, and mediacomponents. A “low process-related impurity composition,” as usedherein, refers to a composition comprising reduced levels ofprocess-related impurities as compared to a composition wherein theimpurities were not reduced. For example, a low process-related impuritycomposition may contain about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, or less process-related impurities. In one embodiment, a lowprocess-related impurity composition is free of process-relatedimpurities or is substantially free of process-related impurities.

In one embodiment, the impurity is a host cell protein. The term “hostcell protein” (HCP), as used herein, is intended to refer to non-proteinof interest proteinaceous impurities derived from host cells, forexample, host cells used to produce the protein of interest.

In one embodiment, the impurity is a host cell nucleic acid. The term“host cell nucleic acids”, as used herein, is intended to refer tonucleic acids derived from host cells, for example, host cells used toproduce the protein of interest.

In a particular embodiment, the impurity is a product-related substance.As used herein, the term “product-related substance” refers to variantsof the protein of interest formed during manufacturing and/or storage ofthe protein of interest. Specific examples of product-related substancesinclude degradants of the protein, truncated forms of the protein, highmolecular weight species, low molecular weight species, fragments of theprotein, modified forms of the protein, including deamidated,isomerized, mismatched S—S linked, oxidized or altered conjugate forms(e.g., glycosylation, phosphorylation), aggregates including dimers andhigher multiples of the protein of interest, and charge variants.

In a particular embodiment, the impurity is an aggregate. As usedherein, the term “aggregate” refers to agglomeration or oligomerizationof two or more individual molecules of the protein of interest to form,for example, dimers, trimers, tetramers, oligomers and other highmolecular weight species. Protein aggregates can be soluble orinsoluble. In a particular embodiment, the aggregate is a multimer ofadalimumab. In a particular embodiment, the aggregate is a dimer ofadalimumab. In another embodiment, the aggregate is a trimer ofadalimumab. In another embodiment, the aggregate is a tetramer ofadalimumab.

In certain embodiments, the sample can comprise more than one type ofaggregate. For example, but not by way of limitation, the nature of theaggregates and total aggregate composition can be identified based onchromatographic residence time. For example, FIG. 1 depicts a sizeexclusion chromatography (SEC) chromatogram used to determine themolecular weight distribution of a sample of adalimumab.

As set forth therein, the total aggregate species associated with theexpression of adalimumab can be divided into multimer 1 (MM1), multimer2 (MM2) and multimer 3 (MM3). In various embodiments, the methods of thepresent invention serve to reduce the levels of one of MM1, MM2 or MM3.In another embodiment, the methods of the present invention serve toreduce the levels of MM1 and MM2. In another embodiment, the methods ofthe present invention serve to reduce the levels of MM1 and MM3. Inanother embodiment, the methods of the present invention serve to reducethe levels of MM2 and MM3. In yet another embodiment, the methods of thepresent invention serve to reduce the levels of MM1, MM2 and MM3.

In one embodiment, the methods of the invention generate a preparationcomprising a protein of interest and having a reduced level ofaggregate. As used herein, a “reduced level of aggregate” refers to acomposition comprising reduced levels of an aggregate as compared to thelevels of the aggregate in the sample prior to purification by themethods of the present invention. In one embodiment, a preparationhaving a reduced level of aggregate is free of the aggregate orsubstantially free of the aggregate. In another embodiment, the methodsof the invention generate a preparation comprising a protein of interestand having a reduced level of total aggregate. As used herein a “reducedlevel of total aggregate” refers to a composition comprising reducedlevels of total aggregate as compared to the levels of the impurity inthe sample prior to purification by the methods of the presentinvention. In one embodiment, a preparation having a reduced level oftotal aggregate is free of aggregates or substantially free of theaggregates.

The present invention is further directed to low aggregate compositionsand methods of generating the same, for example, low aggregatecompositions of adalimumab. The term “low aggregate composition,” asused herein, refers to a composition comprising a protein of interest,wherein the composition contains less than about 15% total aggregates.For example, a low aggregate composition may contain about 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, or lesstotal aggregates. In a particular embodiment, a low aggregatecomposition comprises about 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%,2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%, or less totalaggregates.

In one embodiment, a low aggregate composition of adalimumab cancomprise about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%,2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%, or less of MM1,or 0.0% of MM1. In another embodiment, a low aggregate composition ofadalimumab can comprise about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%,2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%,0.1%, or less of MM2, or 0.0% of MM2. In another embodiment, a lowaggregate composition of adalimumab can comprise about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%,1.2%, 1.1%, 1%, 0.5%, 0.1%, or less of MM3, or 0.0% of MM3.

The term “non-low aggregate composition,” as used herein, refers to acomposition comprising a protein of interest, which contains more thanabout 15% total aggregates. For example, a non-low aggregate compositionmay contain about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,or more total aggregates. In one embodiment, a non-low aggregatecomposition of adalimumab can comprise about 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, or more of MM1. In another embodiment, anon-low aggregate composition or adalimumab can comprise about 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or more of MM2. In anotherembodiment, a non-low aggregate composition or adalimumab can compriseabout 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or more ofMM3.

In one embodiment, a low aggregate composition has improved biologicaland functional properties, including increased efficacy in the treatmentor prevention of a disorder in a subject, e.g., a disorder in which TNFαactivity is detrimental, as compared to a non-low aggregate composition.In one embodiment, the low aggregate composition comprises an anti-TNFαantibody, or antigen-binding portion thereof, such as adalimumab or afragment thereof. For example, in one embodiment, a low aggregatecomposition comprising an antibody, or antigen-binding portion thereof,exhibits increased cartilage penetration, decreased bone erosion, and/orreduced cartilage destruction, as compared to a non-low aggregatecomposition comprising the same antibody or antigen binding portionthereof, when administered to a subject suffering from a disorder inwhich TNFα activity is detrimental.

In another embodiment, a low aggregate composition comprising anantibody, or antigen-binding portion thereof, exhibits increasedprotection against the development of arthritic scores and/orhistopathology scores as compared to a non-low aggregate compositionwhen administered to an animal model of arthritis, e.g., the TNF-Tg197model of arthritis, as described above.

In another embodiment, a low aggregate composition comprising anantibody, or antigen-binding portion thereof, exhibits reduced synovialproliferation, reduced cell infiltration, reduced chondrocyte death,and/or reduced proteoglycan loss as compared to a non-low aggregatecomposition. In another embodiment, a low aggregate compositioncomprising an anti-TNFα antibody, or antigen-binding portion thereof,exhibits increased TNFα affinity as compared to a non-low aggregatecomposition.

In a particular embodiment, the impurity is a fragment of the protein ofinterest. The term “fragment” as used herein refers to any truncatedform of a protein of interest, resulting from, for example, dissociationof a peptide chain, or enzymatic and/or chemical modifications.

In a particular embodiment, the impurity is a charge variant. The term“charge variant”, as used herein, refers to the full complement ofproduct variants including, but not limited to acidic species, and basicspecies (e.g., Lys variants). In certain embodiments, such variants caninclude product aggregates and/or product fragments, to the extent thatsuch aggregation and/or fragmentation results in a product chargevariation as seen in an analytical technique used for that purpose.

As used herein, the terms “acidic species,” “acidic region,” and “AR,”refer to the variants of a protein, e.g., an antibody or antigen-bindingportion thereof, which are characterized by an overall acidic charge.For example, in monoclonal antibody (mAb) preparations, such acidicspecies can be detected by various methods, such as ion exchange, forexample, WCX-10 HPLC (a weak cation exchange chromatography), or IEF(isoelectric focusing). Acidic species of an antibody may include chargevariants, structure variants, and/or fragmentation variants. Exemplarycharge variants include, but are not limited to, deamidation variants,afucosylation variants, methylglyoxal (MGO) variants, glycationvariants, and citric acid variants. Exemplary structure variantsinclude, but are not limited to, glycosylation variants and acetonationvariants. Exemplary fragmentation variants include any truncated proteinspecies from the target molecule due to dissociation of peptide chain,enzymatic and/or chemical modifications, including, but not limited to,Fc and Fab fragments, fragments missing a Fab, fragments missing a heavychain variable domain, C-terminal truncation variants, variants withexcision of N-terminal Asp in the light chain, and variants havingN-terminal truncation of the light chain. Other acidic species variantsinclude variants containing unpaired disulfides, host cell proteins, andhost nucleic acids, chromatographic materials, and media components.

In certain embodiments, a protein composition can comprise more than onetype of acidic species variant. For example, but not by way oflimitation, the total acidic species can be divided based onchromatographic residence time. For example, the total acidic speciesassociated with the expression of adalimumab can be divided into a firstacidic species region (AR1) and a second acidic species region (AR2).

AR1 can comprise, for example, charge variants such as deamidationvariants, MGO modified species, glycation variants, and citric acidvariants, structural variants such as glycosylation variants andacetonation variants, and/or fragmentation variants. Other acidicvariants such as host cells and unknown species may also be present. Inanother embodiment, AR2 can comprise, for example, charge variants suchas glycation variants and deamidation variants. Other acidic variantssuch as host cells and unknown species may also be present.

With respect, in particular, to adalimumab (and antibodies sharingcertain structural characteristics of adalimumab, e.g., one or more CDRand/or heavy and light chain variable regions of adalimumab), AR1 chargevariants can comprise, but are not limited to, deamidation variants,glycation variants, afucosylation variants, MGO variants or citric acidvariants. In one embodiment, deamidation variants result fromdeamidation occurring at asparagine residues comprising Asn393 andAsn329 and at glutamine residues comprising Gln3 and Gln6. In anotherembodiment, the glycation variants result from glycation occurring atLys98 and Lys151. AR1 structure variants can comprise, but are notlimited to, glycosylation variants or acetonation variants. AR1fragmentation variants can comprise Fc and Fab fragments, fragmentsmissing a Fab, fragments missing a heavy chain variable domain,C-terminal truncation variants, variants with excision of N-terminal Aspin the light chain, and variants having N-terminal truncation of thelight chain. AR2 charge variants can comprise, but are not limited to,deamidation variants or glycation variants, wherein the deamidationvariants can result from deamidation occurring at asparagine residuescomprising Asn393 and Asn329 and at glutamine residues comprising Gln3and Gln6, and the glycation variants can result from glycation occurringat Lys98 and Lys151.

Acidic species may also include process-related impurities.

The acidic species may be the result of product preparation (referred toherein as “preparation-derived acidic species”), or the result ofstorage (referred to herein as “storage-derived acidic species”).Preparation-derived acidic species are acidic species that are formedduring the preparation (upstream and/or downstream processing) of theprotein, e.g., the antibody or antigen-binding portion thereof. Forexample, preparation-derived acidic species can be formed during cellculture (“cell culture-derived acidic species”). Storage-derived acidicspecies are acidic species that are not present in the population ofproteins directly after preparation, but are formed while the sample isbeing stored. The type and amount of storage-derived acidic species canvary based on the formulation of the sample. Formation ofstorage-derived acidic species can be partially or completely inhibitedwhen the preparation is stored under particular conditions. For example,an aqueous formulation can be stored at a particular temperature topartially or completely inhibit AR formation. For example, formation orstorage-derived AR can be partially inhibited in an aqueous formulationstored at between about 2° C. and 8° C., and completely inhibited whenstored at −80° C.

In addition, a low AR composition can be lyophilized to partially orcompletely inhibit the formation of storage-derived AR.

The term “low acidic species composition,” as used herein, refers to acomposition comprising an antibody or antigen binding portion thereof,wherein the composition contains less than about 15% acidic species. Forexample, a low acidic species composition may contain about 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, or lessacidic species. In one embodiment, a low acidic species composition cancomprise about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%,2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%, or less of AR1,or 0.0% of AR1. In another embodiment, a low acidic species compositioncan comprise about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2.4%, 2.3%,2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%, or less ofAR2, or 0.0% of AR2. In a preferred embodiment, a low acidic speciescomposition comprises about 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%,2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%, or less acidicspecies. In one embodiment, a low acidic species composition comprisesabout 0.1% or less AR1 and about 3% or less AR2. In another preferredembodiment, a low acidic species composition comprises about 1% or 0.1%or less AR1. In still another preferred embodiment, a low acidic speciescomposition comprises about 3% or less AR2. In another preferredembodiment, the low AR composition comprises about 1.4% or less AR. Forexample, in one embodiment, the composition comprises about 1.4% AR2 andabout 0.0% AR1.

The term “non-low acidic species composition,” as used herein, refers toa composition comprising an antibody or antigen binding portion thereof,which contains more than about 15% acidic species. For example, anon-low acidic species composition may contain about 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, or more acidic species. In oneembodiment, a non-low acidic species composition can comprise about 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or more of AR1. Inanother embodiment, a non-low acidic species composition can compriseabout 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or more ofAR2.

In one embodiment, a low AR composition has improved biological andfunctional properties, including increased efficacy in the treatment orprevention of a disorder in a subject, e.g., a disorder in which TNFαactivity is detrimental, as compared to a non-low acidic speciescomposition. In one embodiment, the low AR composition comprises ananti-TNFα antibody, or antigen-binding portion thereof, such asadalimumab or a fragment thereof. For example, in one embodiment, a lowAR composition comprising an antibody, or antigen-binding portionthereof, exhibits increased cartilage penetration, decreased boneerosion, and/or reduced cartilage destruction, as compared to a non-lowacidic species composition comprising the same antibody or antigenbinding portion thereof, when administered to a subject suffering from adisorder in which TNFα activity is detrimental.

In another embodiment, a low AR composition comprising an antibody, orantigen-binding portion thereof, exhibits increased protection againstthe development of arthritic scores and/or histopathology scores ascompared to a non-low acidic species composition when administered to ananimal model of arthritis, e.g., the TNF-Tg197 model of arthritis, asdescribed above.

In another embodiment, a low AR composition comprising an antibody, orantigen-binding portion thereof, exhibits reduced synovialproliferation, reduced cell infiltration, reduced chondrocyte death,and/or reduced proteoglycan loss as compared to a non-low acidic speciescomposition. In another embodiment, a low AR composition comprising ananti-TNFα antibody, or antigen-binding portion thereof, exhibitsincreased TNFα affinity as compared to a non-low acidic speciescomposition.

In another embodiment, the impurity is a lysine variant species. As usedherein, the term “lysine variant species” refers to an antibody, orantigen-binding portion thereof, comprising heavy chains with eitherzero, one or two C-terminal lysines. For example, the “Lys 0” variantcomprises an antibody, or antigen-binding portion thereof, with heavychains that do not comprise a C-terminal lysine. The “Lys 1” variantcomprises an antibody, or antigen-binding portion thereof, with oneheavy chain that comprises a C-terminal lysine. The “Lys 2” variantcomprises an antibody with both heavy chains comprising a C-terminallysine. Lysine variants can be detected, for example, by weak cationexchange chromatography (WCX) of the expression product of a host cellexpressing the antibody, or antigen-binding portion thereof. Withrespect specifically to adalimumab, three main basic lysine variantspecies have been identified, i.e., Lys 0, Lys 1, and Lys 2.

The term “load buffer”, as used herein refers to a salt solution passedthrough the HIC media upon contacting the sample with the HIC media. Incertain embodiments, the load buffer is passed through the HIC mediasimultaneously or substantially simultaneously with passage of thesample through the HIC media. In certain embodiments, the load buffer iscombined with the sample prior to passage through the HIC media.

The term “flow through fraction”, as used herein refers to the liquidthat passes through without binding the hydrophobic column uponcontacting the sample with the HIC media during the load cycle.According to the methods of the present invention, the flow throughfraction includes protein of interest that does not bind to the HICmedia. The flow through fraction may also include load buffer thatpasses through the HIC media during the load cycle and/or a portion ofthe impurity that does not bind to the HIC media.

The term “wash buffer”, as used herein refers to a salt solution passedthrough the HIC media during the wash cycle.

The term “wash fraction”, as used herein refers to the liquid elutedfrom the column upon washing the HIC media with the wash buffer.According to the methods of the present invention, the wash fractionincludes protein of interest that is released from the HIC media uponexposure to the wash buffer. The wash fraction may also include washbuffer that passes through the HIC media during the wash cycle and/or aportion of the impurity that does not bind to the HIC media.

The term “isocratic”, as used herein, refers to wash and load conditionswhich are identical or vary only slightly in terms of, for example, thenature of the buffer, the salt concentration, the pH, and thetemperature. In particular embodiments, the wash and load conditions aresubstantially the same, for example, the salt concentration and/or thepH of the wash buffer are identical to or are adjusted to within about20%, about 15%, about 10%, or about 5% of the salt concentration, and/orpH of the loading buffer. In a particular embodiment, the wash and loadconditions are identical.

The term “load challenge”, as used herein refers to the total mass ofsample (e.g., protein of interest and at least one protein) loaded ontothe column in chromatography applications or applied to the resin inbatch binding, measured in units of mass of product per unit volume ofresin.

As used herein, the term “dynamic binding capacity” refers to the amountof total protein that binds to the HIC media upon breakthrough of 10% ofthe total protein load.

As used herein, the term “apparent binding capacity” refers to theamount of protein of interest that binds to the HIC media uponbreakthrough of 10% of the total protein load, in reversible HIC bindingapplications.

As used herein, the term “actual binding capacity” refers to the amountof protein of interest that remains bound to the chromatographic mediaunder isocratic wash conditions.

As used herein, the term “equilibrium binding capacity” refers to themaximum amount of total protein that can be bound under certainconditions.

As used herein, the term “partition coefficient” (Kp) refers to theequilibrium ratio of the concentration of protein of interest adsorbedto the HIC media to the concentration of protein of interest in thesolution comprising the unbound protein of interest, under specifiedconditions of pH and solution composition. The partition coefficient Kpcorresponds to the slope of the protein of interest adsorption isothermat very low solution concentrations. Kp can be calculated from the Qmax(maximum capacity of the HIC media for the protein of interest) and Kd(dissociation constant for the HIC media-protein of interestinteraction) as follows: Kp=Q/C=Qmax/Kd.

Protein Purification Protein Purification Generally

The present invention provides a method for producing a preparationincluding a protein of interest, e.g., an antibody, and having a reducedlevel of at least one impurity, e.g., an aggregate, by contacting asample including the protein of interest and at least one impurity, to ahydrophobic interaction chromatography media.

In certain embodiments, the compositions of the present inventioninclude, but are not limited to, a preparation comprising a protein ofinterest having a reduced level of at least one impurity. For example,but not by way of limitation, the present invention is directed topreparations of adalimumab having a reduced level of at least oneimpurity, for example, aggregate. Such preparations having a reducedlevel of at least one impurity address the need for improved productcharacteristics, including, but not limited to, product stability,product safety and product efficacy. In further embodiments,compositions of the present invention include pharmaceuticalcompositions comprising the preparation produced by the methods of theinvention (e.g., protein of interest having a reduced level of the atleast on impurity) and a pharmaceutically acceptable carrier.

In certain embodiments, the purification process of the invention beginsat the separation step when the protein of interest has been producedusing production methods described above and/or by alternativeproduction methods conventional in the art. Once a clarified solution orsample comprising the protein of interest has been obtained, separationof the protein of interest from at least one impurity, such asprocess-related impurities, e.g., other proteins produced by the cell,as well as any product-related substances, e.g., charge variants and/orsize variants (aggregates and fragments), can be performed using a HICseparation step, or a combination of a HIC separation step and one ormore purification techniques, including filtration and/or affinity, ionexchange, and/or mixed mode chromatographic step(s), as outlined herein.

Primary Recovery

In certain embodiments, the initial steps of the purification methods ofthe present invention involve the clarification and primary recovery ofprotein of interest, for example, antibody, following production. Incertain embodiments, the primary recovery will include one or morecentrifugation steps to separate the protein of interest from cells andcell debris. Centrifugation of the protein containing composition can berun at, for example, but not by way of limitation, 7,000×g toapproximately 12,750×g. In the context of large scale purification, suchcentrifugation can occur on-line with a flow rate set to achieve, forexample, but not by way of limitation, a turbidity level of 150 NTU inthe resulting supernatant. Such supernatant can then be collected forfurther purification, or in-line filtered through one or more depthfilters for further clarification of the sample.

In certain embodiments, the primary recovery will include the use of oneor more depth filtration steps to clarify the sample and thereby aid inpurifying the protein of interest in the present invention. In otherembodiments, the primary recovery will include the use of one or moredepth filtration steps post centrifugation to further clarify thesample. Non-limiting examples of depth filters that can be used in thecontext of the instant invention include the Millistak+ XOHC, FOHC,DOHC, A1HC, B1HC depth filters (EMD Millipore), Cuno™ model 30/60ZA,60/90 ZA, VR05, VR07, delipid depth filters (3M Corp.). A 0.2 μm filtersuch as Sartorius's 0.45/0.2 μm Sartopore™ bi-layer or Millipore'sExpress SHR or SHC filter cartridges typically follows the depthfilters.

In certain embodiments, the primary recovery process can also be a pointat which to reduce or inactivate viruses that can be present in thesample. For example, any one or more of a variety of methods of viralreduction/inactivation can be used during the primary recovery phase ofpurification including heat inactivation (pasteurization), pHinactivation, solvent/detergent treatment, UV and γ-ray irradiation andthe addition of certain chemical inactivating agents such asβ-propiolactone or e.g., copper phenanthroline as in U.S. Pat. No.4,534,972. In certain embodiments of the present invention, the sampleis exposed to detergent viral inactivation during the primary recoveryphase. In other embodiments, the sample may be exposed to low pHinactivation during the primary recovery phase.

In those embodiments where viral reduction/inactivation is employed, thesample can be adjusted, as needed, for further purification steps. Forexample, following low pH viral inactivation, the pH of the sample istypically adjusted to a more neutral pH, e.g., from about 4.5 to about8.5, prior to continuing the purification process. Additionally, themixture may be diluted with water for injection (WFI) to obtain adesired conductivity.

Hydrophobic Interaction Chromatography

The instant invention features methods for producing a preparationcomprising a protein of interest (e.g., the anti-TNFα antibodyadalimumab, or a fragment thereof) having a reduced level of at leastone impurity, for example, aggregate, from a sample comprising theprotein of interest and at least one impurity by contacting the samplewith HIC media.

According to the present invention, HIC purification of a protein ofinterest comprises reversible binding of the protein of interest andbinding of one or more impurities through hydrophobic interaction withhydrophobic moieties attached to a solid matrix support (e.g., agarose).The hydrophobic interaction between molecules results from the tendencyof a polar environment to exclude non-polar (i.e., hydrophobic)molecules. HIC relies on this principle of hydrophobicity of molecules(i.e., the tendency of a given protein to bind adsorptively tohydrophobic sites on a hydrophobic adsorbent body) to separatebiomolecules based on their relative strength of interaction with thehydrophobic moieties (see, e.g., U.S. Pat. No. 4,000,098 and U.S. Pat.No. 3,917,527 which are herein incorporated by reference in theirentirety). An advantage of this separation technique is itsnon-denaturing characteristics and the stabilizing effects of saltsolutions used during loading, washing and or eluting.

Hydrophobic interaction chromatography employs the hydrophobicproperties of molecules (e.g., proteins, polypeptides, lipids) toachieve separation of even closely-related molecules. Hydrophobic groupson the molecules interact with hydrophobic groups of the media or themembrane. In certain embodiments, the more hydrophobic a molecule is,the stronger it will interact with the column or the membrane. Thus, HICsteps, such as those disclosed herein, can be used to remove a varietyof impurities, for example, process-related impurities (e.g., DNA) aswell as product-related species (e.g., high and low molecular weightproduct-related species, such as protein aggregates and fragments).

In one aspect, the present invention provides a method for producing apreparation including a protein of interest, e.g., an antibody, andhaving a reduced level of at least one impurity, e.g., an aggregate, by(a) contacting a sample including the protein of interest and at leastone impurity, to a hydrophobic interaction chromatography media, in thepresence of a load buffer such that (i) a portion of the protein ofinterest binds to the hydrophobic interaction chromatography (HIC) mediaand (ii) a substantial portion of the at least one impurity binds to theHIC media; (b) collecting a flow through fraction including the proteinof interest unbound to the HIC media; (c) washing the HIC media with awash buffer that is substantially the same as the load buffer such thata substantial portion of the protein of interest bound to the HIC mediais released from the media; and (d) collecting a wash fraction includingthe protein of interest released from the HIC media, wherein each of theflow through and wash fractions include the protein of interest and havea reduced level of the at least one impurity. In a particularembodiment, the portion of the protein of interest binds to the HICmedia at a Kp of greater than 10. In a particular embodiment, theportion of the protein of interest binds to the HIC media at a Kp ofgreater than 20. In a particular embodiment, the portion of the proteinof interest binds to the HIC media at a Kp of greater than 100.

In another aspect, the present invention provides a method for producinga preparation including a protein of interest, e.g., an antibody, andhaving a reduced level of at least one impurity, e.g., an aggregate, by(a) contacting a sample including the protein of interest and at leastone impurity, to a HIC media, in the presence of a load buffer such that(i) a portion of the protein of interest binds to the HIC media and (ii)a substantial portion of the at least one impurity binds to the HICmedia; collecting a flow through fraction including the protein ofinterest unbound to the HIC media; (c) washing the HIC media with a washbuffer that is substantially the same as the load buffer such that asubstantial portion of the protein of interest bound to the HIC media isreleased from the media; and (d) collecting a wash fraction includingthe protein of interest released from the HIC media, wherein either (i)the substantial portion of the at least one impurity binds to the HICmedia at a Kp greater than 200 and/or (ii) the protein of interest andthe at least on impurity have a Kp ratio less than 1:7; and wherein eachof the flow through and wash fractions include the protein of interestand have a reduced level of the at least one impurity.

In yet another aspect, the present invention provides a method forproducing a preparation including a protein of interest, e.g., anantibody, and having a reduced level of at least one impurity, e.g., anaggregate, by (a) contacting a sample including the protein of interestand at least one impurity, to a HIC media, in the presence of a loadbuffer such that (i) a portion of the protein of interest binds to theHIC media, and (ii) a substantial portion of the at least one impuritybinds to the HIC media; (b) collecting a flow through fraction includingthe protein of interest unbound to the HIC media; (c) washing the HICmedia with a wash buffer that is substantially the same as the loadbuffer such that a substantial portion of the protein of interest boundto the HIC media is released from the media; and (d) collecting a washfraction including the protein of interest released from the HIC media,wherein the K_(d) for the binding of the protein of interest to the HICmedia is less than 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or 2 timesthe K_(d) for the binding of the at least one impurity to the HIC media,and wherein each of the flow through and wash fractions include theprotein of interest and have a reduced level of the at least oneimpurity.

According to the present invention, a portion of the protein of interestreversibly binds to the HIC media while a portion of the protein ofinterest flows through to form a flow through fraction which has areduced level of impurity. The portion of the protein of interest thatbinds to the HIC media binds reversibly in that the bound protein ofinterest may be released therefrom under isocratic conditions, forexample, by use of a wash buffer that is substantially the same as theload buffer. In contrast, a substantial portion of the at least impurityin the sample binds the HIC media upon loading and a substantial portionthereof remains bound upon washing the HIC media with the wash buffer.

The present invention is based, at least in part, on the finding thatsuch reversible binding can be achieved at relatively high bindingstrength. For example, contrary to the teachings of U.S. Pat. No.8,067,182 which teaches weak partitioning binding of a product, i.e., ata Kp of between 0.1 and 20 and less than 10 for HIC, according to themethods of the present invention, the protein of interest may bind athigher Kp levels so as to achieve higher purification and greaterrecovery of the protein of interest. For example, in a particularembodiment, the protein of interest binds to the HIC media at a Kp ofgreater than 10, 15, 20, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 220, 250, 300, 400 or 500. In oneembodiment, the protein of interest binds to the HIC media at a Kpgreater than 10. In another embodiment, the protein of interest binds tothe HIC media at a Kp greater than 20. In another embodiment, theprotein of interest binds to the HIC media at a Kp of greater than 90.

According to the present invention, the impurity binds at a higherstrength and, thus to a greater degree to the HIC media, therebyallowing for selective release of the bound protein of interest uponwash. For example, in particular embodiments, the at least one impuritybinds to the HIC media at a Kp of greater than 200, greater than 250,greater than 300, greater than 400, greater than 500, greater than 600,greater than 700, greater than 800, greater than 900, greater than 1000,or greater than 2000. In a specific embodiment, the at least oneimpurity binds to the HIC media at a Kp of greater than 600.

In a further embodiment, the protein of interest and the at least oneimpurity have a Kp ratio less than 1:10, less than 1:9, less than 1:8,less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than1:3 or less than 1:2. In a specific embodiment, the protein of interestand the at least one impurity have a Kp ratio less than 1:7.

The relative strength of binding may also be assessed by determiningK_(d), the dissociation constant for the media-protein of interestinteraction, or the media-impurity interaction. In one embodiment of theinvention, the K_(d) for the binding of the protein of interest to theHIC media is at least about 0.2, at least about 0.3, at least about 0.4,at least about 0.5, at least about 0.6, at least about 0.7, or at leastabout 0.8. In a preferred embodiment, the K_(d) for the binding of theprotein of interest to the HIC media is at least about 0.4.

In another embodiment of the invention, the K_(d) of the binding of theat least one impurity to the HIC media is less than or equal to about0.001, about 0.005, about 0.01, about 0.02, about 0.05, about 0.1, about0.15, about 0.2, about 0.3, about 0.4, about 0.5, or about 1.0. In aparticular embodiment the K_(d) for the at least one impurity is lessthan or equal to about 0.01.

In another embodiment of the invention, the K_(d) for the binding of theprotein of interest to the HIC media is less than 60, 50, 45, 40, 35,30, 25, 20, 15, 10, 5 or 2 times the K_(d) for the binding of the atleast one impurity to the HIC media.

The relative binding capacity of the protein of interest may also beassessed by determining Qmax, the maximum capacity of the media for theprotein of interest, or for the at least one impurity. In one embodimentof the invention, the protein of interest has a Qmax of at least about20, at least about 30, at least about 40, at least about 50, at leastabout 60, at least about 100, at least about 250, or at least about 500.In a preferred embodiment, the protein of interest has a Qmax of atleast about 40.

In another embodiment, the at least one impurity has a Qmax of at leastabout 2, at least about 5, at least about 10, at least about 20, atleast about 30, at least about 40, at least about 50, at least about 75,or at least about 100. In a preferred embodiment, the at least oneimpurity has a Qmax of at least about 5.

In performing the HIC separation, the sample is contacted with the HICmedia, e.g., using a batch purification technique or using a column ormembrane chromatography or monolithic material (referred to as HIC mediaor resin). For example, in the context of chromatographic separation, achromatographic apparatus, commonly cylindrical in shape, is employed tocontain the chromatographic support media (e.g., HIC media) prepared inan appropriate buffer solution. Once the chromatographic material isadded to the chromatographic apparatus, a sample containing the proteinof interest, e.g., an antibody, and the protein of interest is contactedto the chromatographic material in the presence of a loading buffer toallow binding of a portion of the protein of interest and a substantialportion of the impurity to the HIC media. A portion of the protein ofinterest in the sample binds to the HIC media while a portion of theprotein interest flows through, forming a flow through fraction having areduced level of impurity which is collected.

In one embodiment, the portion of the protein of interest that binds tothe HIC media is at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95% or at leastabout 98% of the amount of the protein of interest in the sample.

Alternatively or in combination, the substantial portion of the at leastone impurity that binds to the HIC media is at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98% or about 100% of the level of the at least one impurity in thesample.

Alternatively or in combination, the portion of the protein of interestthat flows through without binding to the HIC media is at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95% or at least about 98% of the amount of theprotein of interest in the sample.

The media is then subjected to a wash buffer, thereby allowing for aportion of the bound protein of interest to release from the HIC mediain a wash fraction which is collected, while a substantial portion ofthe impurity remains bound to the HIC media. After loading, the columncan be regenerated with water and cleaned with caustic solution toremove the bound impurities before next use.

In one embodiment, the substantial portion of the protein of interestreleased from the HIC media upon washing with the wash buffer is atleast about at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, or about 100% of the amount of protein of interest bound tothe HIC media.

Alternatively or in combination, the substantial portion of the impuritythat remains bound to the HIC media is at least about 50%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 98% or about 100% of the impurity bound to theHIC media during the load cycle.

In order to achieve the desired reversible binding of the protein ofinterest and the comparable strong binding of the at least one impurity,appropriate selection of resin, buffer, concentration, pH and sampleload is required. Techniques to identify optimal conditions forachieving such desired binding profile are set forth in the Examplesbelow.

Hydrophobic interactions are strongest at high salt concentration (andhence the ionic strength of the anion and cation components). Adsorptionof the protein of interest to a HIC column is favored by high saltconcentrations, but the actual concentrations can vary over a wide rangedepending on the nature of the protein of interest, salt type and theparticular HIC ligand chosen. In various embodiments, the saltconcentration may be in the range of, for example, about 50 mM to about5000 mM, about 100 mM to about 4000 mM, about 1000 mM to about 4000 mM,about 50 mM to about 2000 mM, depending, in part, on the salt type andHIC adsorbent. In one embodiment the salt concentration is about 50 mM,about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about80 mM, about 85 mM, about 90 mM, about 100 mM, about 200 mM, about 300mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800mM, about 900 mM, about 1000 mM, about 1200 mM, about 1400 mM, about1600 mM, about 1800 mM or about 2000 mM.

Various ions can be arranged in a so-called soluphobic series dependingon whether they promote hydrophobic interactions (salting-out effects)or disrupt the structure of water (chaotropic effect) and lead to theweakening of the hydrophobic interaction. Cations are ranked in terms ofincreasing salting out effect as Ba²⁺; Ca²⁺; Mg²⁺; Li⁺; Cs⁺; Na⁺; K⁺;Rb⁺; NH₄ ⁺, while anions may be ranked in terms of increasing chaotropiceffect as PO₄ ³⁻; SO₄ ²⁻; CH₃CO₃ ⁻; Cl⁻; Br⁻; NO₃ ⁻; ClO₄ ⁻; I⁻; SCN⁻.

In certain embodiments, the anionic part of the salt is chosen fromamong sulfate, citrate, chloride, or a mixture thereof. In certainembodiments, the cationic part of the salt is chosen from amongammonium, sodium, potassium, or a mixture thereof. In general, Na⁺, K⁺or NH₄ ⁺ sulfates effectively promote ligand-protein interaction in HIC.Salts may be formulated that influence the strength of the interactionas given by the following relationship:(NH₄)₂SO₄>Na₂SO₄>NaCl>NH₄Cl>NaBr>NaSCN. In general, salt concentrationsof between about 0.75 and about 2 M ammonium sulfate or between about 1and 4 M NaCl are useful. In another embodiment, the load buffer and thewash buffer comprise a salt of the Hofmeister series or lyotropic seriesof salts.

In one embodiment, the load buffer and the wash buffer comprise asulfate salt, a citrate salt, or a combination thereof. In anotherembodiment, the sulfate salt in ammonium sulfate. In another embodiment,the sulfate salt is a sodium sulfate. In yet another embodiment, thecitrate salt is sodium citrate. In certain embodiments, the load and/orwash buffer may be comprised of at least 2 salts.

In certain embodiments, the HIC adsorbent material is composed of achromatographic backbone with pendant hydrophobic interaction ligands.For example, but not by way of limitation, the HIC media can be composedof convective membrane media with pendent hydrophobic interactionligands, convective monolithic media with pendent hydrophobicinteraction ligands, and/or convective filter media with embedded mediacontaining the pendant hydrophobic interaction ligands.

In certain embodiments, the HIC adsorbent material can comprise a basematrix (e.g., derivatives of cellulose, polystyrene, synthetic polyamino acids, synthetic polyacrylamide gels, cross-linked dextran,cross-linked agarose, synthetic copolymer material or even a glasssurface) to which hydrophobic ligands (e.g., alkyl, aryl andcombinations thereof) are coupled or covalently attached usingdifunctional linking groups such as —NH—, —S—, —COO—, etc. Thehydrophobic ligand may be terminated in a hydrogen but can alsoterminate in a functional group such as, for example, NH₂, SO₃H, PO₄H₂,SH, imidazoles, phenolic groups or non-ionic radicals such as OH andCONH₂. In one embodiment, the HIC media comprises at least onehydrophobic ligand. In another embodiment, the hydrophobic ligand isselected from the group consisting of butyl, hexyl, phenyl, octyl, orpolypropylene glycol ligands.

One, non-limiting, example of a suitable HIC media comprises an agarosemedia or a membrane functionalized with phenyl groups (e.g., a PhenylSepharose™ from GE Healthcare or a Phenyl Membrane from Sartorius). ManyHIC medias are available commercially. Examples include, but are notlimited to, Tosoh Hexyl, CaptoPhenyl, Phenyl Sepharose™ 6 Fast Flow withlow or high substitution, Phenyl Sepharose™ High Performance, OctylSepharose™ High Performance (GE Healthcare); Fractogel™ EMD Propyl orFractogel™ EMD Phenyl (E. Merck, Germany); Macro-Prep™ Methyl orMacro-Prep™ t-Butyl columns (Bio-Rad, California); WP HI-Propyl (C3)™(J. T. Baker, New Jersey); Toyopearl™ ether, phenyl or butyl (TosoHaas,PA); ToyoScreen PPG, ToyoScreen Phenyl, ToyoScreen Butyl, and ToyoScreenHexyl are a rigid methacrylic polymer bead. GE HiScreen Butyl FF andHiScreen Octyl FF are high flow agarose based beads.

In one embodiment, the HIC media has a dynamic binding capacity of atleast about 2 g, at least about 5 g, at least about 10 g, at least about20 g, at least about 30 g, at least about 40 g, at least about 50 g, atleast about 60 g, at least about 70 g, at least about 80 g, at leastabout 90 g, at least about 100 g, or at least about 200 g of sample perone liter of media.

Because the pH selected for any particular purification process must becompatible with protein stability and activity, particular pH conditionsmay be specific for each application. A high or low pH may serve toweaken hydrophobic interactions and retention of proteins changes.

The pH of the HIC purification process is dependent, in part, on the pHof the buffers used to load, equilibrate and or wash the chromatographicresin or media.

Accordingly, in one embodiment, the pH of any of the buffers is betweenabout 4.0 and 8.5. In a further embodiment, the pH of any of the buffersis between about 5.0 and 7.0. In one embodiment, the pH of any of thebuffers may be about 4.0, about 4.5, about 5.0, about 5.5, about 6.0,about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5. In a preferredembodiment the pH of any of the buffers is 5.0. In a related embodimentthe pH of any of the buffers is 5.6. In yet another embodiment, the pHof any of the buffers is 7.0.

In certain embodiments, the load challenge of the sample comprising theprotein of interest and at least one impurity is adjusted to a totalprotein load to the column of between about 50 and 1000 g/L, or betweenabout 250 and 700 g/L, or between about 350 and 500 g/L of HIC media. Incertain embodiments, the protein concentration of the load challenge isadjusted to a total protein concentration of about 0.5 and 50 g/L, orbetween about 1 and 20 g/L, or between about 3 and 10 g/L. In oneembodiment the load challenge is about 50 g, about 100 g, about 150 g,about 200 g, about 250 g, about 300 g, about 350 g, about 400 g, about450 g, about 500 g, about 550 g, about 600 g, about 650 g, about 700 g,about 750 g, about 800 g, about 850 g, about 900 g, about 950 g, orabout 1000 g of sample per one liter of HIC media. In a particularembodiment, the load challenge of the sample is 200 g/L. In anotherembodiment, the load challenge is 350 g/L. In yet another embodiment,load challenge of the sample is 500 g/L. In yet another embodiment, theload challenge of the sample is 700 g/L.

In another embodiment, the load challenge for the impurity alone isabout 0.1 g, about 0.2 g, about 0.3 g, about 0.4 g, about 0.5 g, about0.6 g, about 0.7 g, about 0.8 g, about 0.9 g, about 1.0 g, about 1.5 g,about 2.0 g, about 2.5 g, about 3.0 g, about 3.5 g, about 4.0 g, about4.5 g, or about 5.0 g of the at least one impurity per one liter of HICmedia.

In certain embodiments, impurity (e.g., aggregate) concentration ismeasured and used as a parameter for controlling impurity clearance inthe present invention. For example, but not by way of limitation, thedata presented in the Examples below, demonstrates that impurityconcentration influences the impurity reduction by hydrophobicinteraction chromatography. Thus, in certain embodiments, the at leastone impurity concentration is adjusted from about 0.5 to 0.1 g/L, toabout 0.1 to 0.05 g/L or to below 0.05 g/L. In another embodiment, theat least one impurity contacting the HIC media has a concentration ofabout 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06,about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.2,about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about0.9, about 1.0, about 2.0, about 3.0, about 4.0 or about 5.0 g/L.

In certain embodiments, protein of interest concentration (e.g.,antibody monomer) is measured and used as a parameter for controllingimpurity (e.g., aggregate) clearance in the present invention. Forexample, but not by way of limitation, the data presented in theExamples below demonstrates that control of the concentration of theprotein of interest can be used to achieve improved impurity clearance.Thus, in certain embodiments, the protein of interest concentration isadjusted from about 15 to 8 g/L, to about 8 to 4 g/L or to below 4 g/L.In another embodiment, the protein of interest contacting the HIC mediahas a concentration of less than about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 15, about20, about 25, about 30, about 35, about 40 about 45, about 50, or about55 g/L.

In certain embodiments, protein of interest (e.g., antibody monomer) andimpurity (e.g., aggregate) concentration is measured and used as aparameter for controlling impurity clearance in the present invention.For example, but not by way of limitation, the data presented in theExamples demonstrates that control of the protein of interest andmonomer concentrations within certain ranges can be used to achieveimproved impurity clearance. Thus, in certain embodiments, the proteinof interest concentration is adjusted from about 20 to 15 g/L, about 15to 8 g/L or to below 4 g/L and the impurity concentration is adjusted to0.5 to 0.1 g/L, about 0.1 to 0.05 g/L or to below 0.05 g/L to achieveimpurity reduction in the present invention. In another embodiment, theprotein of interest contacting the HIC media has a concentration ofabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 15, about 20, about 25, about 30, about 35,about 40 about 45, or about 55 g/L and the at least one impuritycontacting the HIC media has a concentration of less than about 0.01,about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07,about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about 0.3,about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, orabout 1.0 g/L.

In another embodiment, the sample contacting the HIC media has aconcentration of about 1, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 15, about 20, about 25, about30, about 35, about 40 about 45, or about 55 g/L.

In one embodiment, the at least one impurity is an aggregate of theprotein of interest, for example, selected from the group consisting ofa dimer, a trimer, a tetramer, an oligomer and other high molecularweight species. In a particular embodiment, the protein of interest isadalimumab and the at least one impurity is an aggregate of adalimumab.For example, the aggregate may be selected from the group consisting ofmultimer 1, multimer 2 and multimer 3.

In another embodiment, the impurity is a process-related impurity or aproduct-related substance. For example, the impurity may be aprocess-related impurity selected from the group consisting of a hostcell protein, a host cell nucleic acid, a media component, and achromatographic material. Alternatively, the impurity may be aproduct-related substance selected from the group consisting of a chargevariant, an aggregate of the protein of interest, a fragment of theprotein of interest and a modified protein.

In a particular embodiment the impurity is an acidic or basic variant,for example, of adalimumab. In a particular embodiment, the basicvariant is a lysine variant species, for example, an antibody, orantigen-binding portion thereof, having heavy chains with either zero,one or two C-terminal lysines. In another embodiment, the impurity is anacidic species (AR), for example, selected from the group consisting ofa charge variant, a structure variant, a fragmentation variant, aprocess-related impurity and a product-related impurity. In a particularembodiment, the acidic species is AR1 and the charge variant is adeamidation variant, a glycation variant, an afucosylation variant, aMGO variant and/or a citric acid variant. In another embodiment, theacidic species is AR1 and the structure variant is a glycosylationvariant and/or an acetonation variant. In yet another embodiment, theacidic species is AR1 and the fragmentation variant is a Fab fragmentvariant, a C-terminal truncation variant or a variant missing a heavychain variable domain. In yet a further embodiment, the acidic speciesis AR2 and the charge variant comprises a deamidation variant and/orglycation variant.

In a particular embodiment, the impurity is a fragment such as an Fc ora Fab fragment. In another embodiment, the impurity is a modifiedprotein such as a deamidated protein or glycosylated protein.

In certain embodiments, HIC chromatographic fractions are collectedduring the load and/or wash cycles and are combined after appropriateanalysis to provide a protein preparation that contains the reducedlevel of impurities. In certain embodiments, the flow through fractionis combined with certain wash fractions to improve the yield of theprocess while still achieving the desired, e.g., reduced level ofimpurities in the preparation.

Additionally, the flow through or wash fractions, or combination thereofmay be contacted with HIC media again to further purify the sample. Invarious embodiments, the method may be repeated at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15 or 20 times.

In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,Fluorescence, Raman may be used to monitor levels of impurities such asaggregates and low molecular weight variants (e.g., fragments of theprotein of interest) in an on-line, at-line or in-line mode, which canthen be used to control the level of aggregates in the pooled materialcollected from the HIC methods of the present invention. In certainembodiments, on-line, at-line or in-line monitoring methods can be usedeither on the wash line of the chromatography step or in the collectionvessel, to enable achievement of the desired product quality/recovery.In certain embodiments, the UV signal can be used as a surrogate toachieve an appropriate product quality/recovery, wherein the UV signalcan be processed appropriately, including, but not limited to, suchprocessing techniques as integration, differentiation, moving average,such that normal process variability can be addressed and the targetproduct quality can be achieved. In certain embodiments, suchmeasurements can be combined with in-line dilution methods such that ionconcentration/conductivity of the load/wash can be controlled byfeedback, thereby facilitating product quality control.

In one embodiment, the reduced level of the at least one impurity of theflow through fractions and/or the wash fractions is at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98% or about 100% of the amount of the at least onimpurity, e.g., aggregate or host cell protein in the sample.

In another embodiment, the impurity is a host cell protein and isreduced by at least 0.25, at least 0.5, at least 0.75, at least 1.0, atleast 1.25, at least 1.5, at least 1.75, at least 2.0, or at least 5.0LFR.

In another embodiment, the accumulative aggregate reduction of the atleast one impurity in any one flow through fraction and/or wash fractioncollected during the preparation is at least about 0.1%, at least about0.2%, at least about 0.5%, at least about 1.0%, at least about 2.0%, atleast about 3.0%, at least about 4.0%, at least about 5.0%, at leastabout 10%, at least about 20%, or at least about 50%.

In another embodiment, the accumulative aggregate reduction of the atleast one impurity in the flow through fraction and the wash fractionsis at least about 0.1%, at least about 0.2%, at least about 0.5%, atleast about 1.0%, at least about 2.0%, at least about 3.0%, at leastabout 4.0%, at least about 5.0%, at least about 10.0%, or at least about20.0%.

In another embodiment, the accumulative yield of the protein of interestin the flow through fraction and in the wash fraction is at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%.

In yet another embodiment, the accumulative yield of the protein ofinterest in any one flow through fraction or wash fraction is at leastabout 4%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85, at least about 90%, at least about95% or about 100%.

Complementary Purification Techniques

In certain embodiments, a combination of HIC and at least one of AEX(anion exchange chromatography) and CEX (cation exchange chromatography)and MM (mixed-mode chromatography) methods can be used to preparepreparations of protein of interest having a reduced level of impurity,including certain embodiments where one technology is used in acomplementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by a particularly technology, suchthat the combination provides the desired final composition/productquality. In certain embodiments, such combinations include the use ofadditional intervening chromatography, filtration, pH adjustment, UF/DF(ultrafiltration/diafiltration) steps so as to achieve the desiredproduct quality, ion concentration, and/or viral reduction.

Affinity Chromatography

In certain embodiments, a precursor sample is subjected to affinitychromatography to purify the protein of interest, prior to the methodsof the present invention. Alternatively or in addition, the wash and/orflow through fractions generated by the methods of the present inventioncan be subjected to affinity chromatography to further purify theprotein of interest. As noted above, certain embodiments of the presentinvention will employ one or more affinity chromatography steps prior tothe HIC purification step, while others will employ an affinitychromatography step after or both before and after the HIC purificationstep. In certain embodiments, the affinity chromatography media is aProtein A, G, A/G, or L media, although alternative affinitychromatography medias are known in the art. There are a variety ofcommercial sources for Protein A media. Suitable medias include, but arenot limited to, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelectXtra, rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra,and ProSep Ultra Plus from EMD Millipore, MapCapture from LifeTechnologies.

In certain embodiments, the Protein A column can be equilibrated with asuitable buffer prior to sample loading. Following the loading of thecolumn, the column can be washed one or multiple times using a suitablesets of buffers. The Protein A column can then be eluted using anappropriate elution buffer. The eluate can be monitored using techniqueswell known to those skilled in the art. The eluate fractions of interestcan be collected and then prepared for further processing.

The Protein A eluate may be subject to a viral inactivation step eitherby detergent or low pH, provided this step is not performed prior to theProtein A capture operation. A proper detergent concentration or pH andtime can be selected to obtain desired viral inactivation results. Afterviral inactivation, the Protein A eluate is usually pH and/orconductivity adjusted for subsequent purification steps.

The Protein A eluate may be subjected to filtration through a depthfilter to remove turbidity and/or various impurities from the antibodyof interest prior to additional chromatographic polishing steps.Examples of depth filters include, but are not limited to, Millistak+XOHC, FOHC, DOHC, AlHC, and B1HC Pod filters (EMD Millipore), or ZetaPlus 30ZA/60ZA, 60ZA/90ZA, delipid, VR07, and VRO5 filters (3M). TheProtein A eluate pool may need to be conditioned to proper pH andconductivity to obtain desired impurity removal and product recoveryfrom the depth filtration step.

Ion Exchange Chromatography

In certain embodiments, a precursor sample is subjected to ion exchangechromatography to purify the protein of interest, prior to the methodsof the present invention. Alternatively or in addition, the wash and/orflow through fractions generated by the methods of the present inventioncan be subjected to ion exchange chromatography to further purify theprotein of interest. As noted above, certain embodiments of the presentinvention will employ one or more ion exchange chromatography stepsprior to the HIC purification step, while others will employ an ionexchange chromatography step after or both before and after the HICpurification step.

As used herein, ion exchange separations includes any method by whichtwo substances are separated based on the difference in their respectiveionic charges, either on the protein of interest and/or chromatographicmaterial as a whole or locally on specific regions of the protein ofinterest and/or chromatographic material, and thus can employ eithercationic exchange material or anionic exchange material.

The use of a cationic exchange material versus an anionic exchangematerial is based on the local charges of the protein of interest in agiven solution. Therefore, it is within the scope of this invention toemploy an anionic exchange step prior to the use of a HIC step, or acationic exchange step prior to the use of an HIC step. Furthermore, itis within the scope of this invention to employ only a cationic exchangestep, only an anionic exchange step, or any serial combination of thetwo either prior to or subsequent to the HIC step.

In performing the separation, the sample containing the protein ofinterest (e.g., an antibody or antigen-binding fragment thereof) can becontacted with the ion exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique, as described above in connection with HIC.

Ion exchange chromatography separates molecules based on differencesbetween the local charges of the proteins of interest and the localcharges of the chromatographic material. A packed ion-exchangechromatography column or an ion-exchange membrane device can be operatedin a bind-elute mode, a flow-through, or a hybrid mode. After washingthe column or the membrane device with the equilibration buffer oranother buffer with different pH and/or conductivity, the productrecovery is achieved by increasing the ionic strength (i.e.,conductivity) of the elution buffer to compete with the solute for thecharged sites of the ion exchange matrix. Changing the pH and therebyaltering the charge of the solute is another way to achieve elution ofthe solute. The change in conductivity or pH may be gradual (gradientelution) or stepwise (step elution). The column is then regeneratedbefore next use.

Anionic or cationic substituents may be attached to matrices in order toform anionic or cationic supports for chromatography. Non-limitingexamples of anionic exchange substituents include diethylaminoethyl(DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups.Cationic substituents include carboxymethyl (CM), sulfoethyl (SE),sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulose ionexchange medias such as DE23™, DE32™, DE52™, CM-23™, CM-32™, and CM-52™are available from Whatman Ltd. Maidstone, Kent, U.K. SEPHADEX®-basedand -locross-linked ion exchangers are also known. For example, DEAE-,QAE-, CM-, and SP-SEPHADEX® and DEAE-, Q-, CM- and S-SEPHAROSE® andSEPHAROSE® Fast Flow, and Capto™ S are all available from GE Healthcare.Further, both DEAE and CM derivitized ethylene glycol-methacrylatecopolymer such as TOYOPEARL™ DEAE-650S or M and TOYOPEARL™ CM-650S or Mare available from Toso Haas Co., Philadelphia, Pa., or Nuvia S andUNOSphere™ S from BioRad, Hercules, Calif., Eshmuno® S from EMDMillipore, Billerica, Calif.

Mixed Mode Chromatography

In certain embodiments, a precursor sample is subjected to mixed modechromatography to purify the protein of interest, prior to the HICmethods of the present invention. Alternatively or in addition, the washand/or flow through fractions generated by the methods of the presentinvention can be subjected to mixed mode chromatography to furtherpurify the protein of interest. As noted above, certain embodiments ofthe present invention will employ one or more mixed mode chromatographysteps prior to the HIC purification step, while others will employ amixed mode chromatography step after or both before and after the HICpurification step.

Mixed mode chromatography is chromatography that utilizes a mixed modemedia, such as, but not limited to CaptoAdhere available from GEHealthcare. Such a media comprises a mixed mode chromatography ligand.In certain embodiments, such a ligand refers to a ligand that is capableof providing at least two different, but co-operative, sites whichinteract with the substance to be bound. One of these sites gives anattractive type of charge-charge interaction between the ligand and theprotein of interest. The other site typically gives electronacceptor-donor interaction and/or hydrophobic and/or hydrophilicinteractions. Electron donor-acceptor interactions include interactionssuch as hydrogen-bonding, π-π, cation-π, charge transfer, dipole-dipole,induced dipole etc. The mixed mode functionality can give a differentselectivity compared to traditional anion exchangers. For example,CaptoAdhere is designed for post-Protein A purification of monoclonalantibodies, where removal of leached Protein A, aggregates, host cellproteins, nucleic acids and viruses from monoclonal antibodies isperformed in flow-through mode (the antibodies pass directly through thecolumn while the contaminants are adsorbed). Mixed mode chromatographyligands are also known as “multimodal” chromatography ligands.

In certain embodiments, the mixed mode chromatography media is comprisedof mixed mode ligands coupled to an organic or inorganic support,sometimes denoted a base matrix, directly or via a spacer. The supportmay be in the form of particles, such as essentially sphericalparticles, a monolith, filter, membrane, surface, capillaries, etc. Incertain embodiments, the support is prepared from a native polymer, suchas cross-linked carbohydrate material, such as agarose, agar, cellulose,dextran, chitosan, konjac, carrageenan, gellan, alginate etc. To obtainhigh adsorption capacities, the support can be porous, and ligands arethen coupled to the external surfaces as well as to the pore surfaces.Such native polymer supports can be prepared according to standardmethods, such as inverse suspension gelation (S Hjerten: Biochim BiophysActa 79(2), 393-398 (1964). Alternatively, the support can be preparedfrom a synthetic polymer, such as cross-linked synthetic polymers, e.g.styrene or styrene derivatives, divinylbenzene, acrylamides, acrylateesters, methacrylate esters, vinyl esters, vinyl amides etc. Suchsynthetic polymers can be produced according to standard methods, seee.g. “Styrene based polymer supports developed by suspensionpolymerization” (R Arshady: Chimica e L′Industria 70(9), 70-75 (1988)).Porous native or synthetic polymer supports are also available fromcommercial sources, such as Amersham Biosciences, Uppsala, Sweden.

Viral Filtration

In certain embodiments, a precursor sample is subjected to viralfiltration to purify the protein of interest, prior to the HIC methodsof the present invention. Alternatively or in addition, the wash and/orflow through fractions generated by the methods of the present inventioncan be subjected to viral filtration to further purify the protein ofinterest. As noted above, certain embodiments of the present inventionwill employ one or more viral filtration steps prior to the HICpurification step, while others will employ viral filtration after orboth before and after the HIC purification step.

Viral filtration is a dedicated viral reduction step in the entirepurification process. This step is usually performed as a postchromatographic polishing step. Viral reduction can be achieved via theuse of suitable filters including, but not limited to, Planova 20N™, 50N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMDMillipore, ViroSart CPV from Sartorius, or Ultipor DV20 or DV50™ filterfrom Pall Corporation. It will be apparent to one of ordinary skill inthe art to select a suitable filter to obtain desired filtrationperformance.

Ultrafiltration/Diafiltration

In certain embodiments, a precursor sample is subjected toultrafiltration and/or diafiltration to purify the protein of interest,prior to the HIC methods of the present invention. Alternatively or inaddition, the wash and/or flow through fractions generated by themethods of the present invention can be subjected to ultrafiltrationand/or diafiltration to further purify the protein of interest. As notedabove, certain embodiments of the present invention will employ one ormore ultrafiltration and/or diafiltration steps prior to the HICpurification step, while others will employ ultrafiltration and/ordiafiltration after or both before and after the HIC purification step.

Ultrafiltration is described in detail in: Microfiltration andUltrafiltration: Principles and Applications, L. Zeman and A. Zydney(Marcel Dekker, Inc., New York, N.Y., 1996); and in: UltrafiltrationHandbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No.87762-456-9). A preferred filtration process is Tangential FlowFiltration as described in the Millipore catalogue entitled“Pharmaceutical Process Filtration Catalogue” pp. 177-202 (Bedford,Mass., 1995/96). Ultrafiltration is generally considered to meanfiltration using filters with a pore size of smaller than 0.1 μm. Byemploying filters having such small pore size, the volume of the samplecan be reduced through permeation of the sample buffer through thefilter while antibodies are retained behind the filter.

Diafiltration is a method of using ultrafilters to remove and exchangesalts, sugars, and non-aqueous solvents, to separate free from boundspecies, to remove low molecular-weight material, and/or to cause therapid change of ionic and/or pH environments. Microsolutes are removedmost efficiently by adding solvent to the solution being ultrafilteredat a rate approximately equal to the ultratfiltration rate. This washesmicrospecies from the solution at a constant volume, effectivelypurifying the retained antibody. In certain embodiments of the presentinvention, a diafiltration step is employed to exchange the variousbuffers used in connection with the instant invention, optionally priorto further chromatography or other purification steps, as well as toremove impurities from the antibody preparations.

Exemplary Purification Strategies

In certain embodiments, primary recovery can proceed by sequentiallyemploying pH reduction, centrifugation, and filtration steps to removecells and cell debris (including HCPs) from the production bioreactorharvest.

Additionally, the HIC methodology as described herein is utilized tofurther purify the protein of interest. As set forth herein, suchmethods involve (a) contacting a sample including the protein ofinterest and at least one impurity, to a hydrophobic interactionchromatography (HIC) media, in the presence of a load buffer such that(i) a portion of the protein of interest binds to the HIC media, forexample, at a Kp of at least 10, 20 or 100 and (ii) a substantialportion of the at least one impurity binds to the HIC media; (b)collecting a flow through fraction including the protein of interestunbound to the HIC media; (c) washing the HIC media with a wash bufferthat is substantially the same as the load buffer such that asubstantial portion of the protein of interest bound to the HIC media isreleased from the media; and (d) collecting a wash fraction includingthe protein of interest released from the HIC media, wherein each of theflow through and wash fractions include the protein of interest and havea reduced level of the at least one impurity.

Examples of buffers that can be used in the context of both the loadingand wash steps of the present invention include, but are not limited to,the following: about 0.1 M to about 0.6 M sodium citrate (NaCit), pH5.6; or about 0.5 M to about 1.1 M ammonium sulfate (AmSO₄), pH 7.0 aswell as buffers substantially the same, in that any differences resultin insubstantial changes to the binding of impurities, but do notsubstantially affect the ability to wash and release antibody product.Such buffers can span a range of varying “hydrophobicities” based on therationales discussed in above.

In certain embodiments, the HIC media employed in the HIC step isCaptoPhenyl (GE) resin. In certain embodiments, the CaptoPhenyl (GE)resin is buffer exchanged into 0.4 M sodium citrate (NaCit), pH 5.6, andthen distributed in 100 □L aliquots into microcentrifuge tubes. Eachtube is then challenged with 2 mL of antibody produce source material,e.g., a partially purified cell culture harvest sample, in 0.4 M NaCit,pH 5.6, at a range of concentrations from 0.5-15.0 mg/mL and incubatedfor 3 hours at room temperature with mixing. The resin is allowed tosettle and the supernatant removed and replaced with 1 mL of fresh 0.4 MNaCit, pH 5.6, buffer and incubated for 2 hours at room temperature withmixing. This step was repeated one more time.

In alternative embodiments, the CaptoPhenyl (GE) HIC resin can be packedin 1.0 cm×10.0 cm (OmniFit) columns. Antibody product HIC-load can beprepared by diluting the source material, e.g., a partially purifiedcell culture harvest sample, with a 1.2 M stock solution of sodiumcitrate (NaCit), pH 5.6, to final concentration in the range of 0.3 to0.5 M NaCit, pH 5.6. CaptoPhenyl columns can then be equilibrated with 7column volumes (CVs) of a NaCit buffer, pH 5.6, corresponding to theload concentration. The antibody product solution can then be loaded tothe column in the range of 200-500 g/L, after which the column is washedwith 20 CVs of the wash buffer. The column can then be regenerated (3CVs f 25 mM sodium phosphate/20% (v/v) isopropyl alcohol, pH 6.5),cleaned in place (3 CVs 1M NaOH, 60 min hold), and stored (5 CVs of 25mM sodium phosphate/20% (v/v) isopropyl alcohol, pH 6.5). The releasedsolution from the column can be fractionated during the entire run andused to monitor the breakthrough of both the protein of interest, e.g.,antibody product monomer, as well as impurities, e.g., aggregates andhost cell protein (HCP).

Such HIC purification steps can be preceded by affinity chromatography,for example, but not limited to, the use of Protein A-base affinitychromatography. There are several commercial sources for Protein Amedia. One suitable media is MabSelect™ from GE Healthcare. An exampleof a suitable column packed with MabSelect™ is a column about 1.0 cmdiameter×about 21.6 cm long (˜17 mL bed volume). This size column can beused for bench scale. This can be compared with other columns used forscale ups. For example, a 20 cm×21 cm column whose bed volume is about6.6 L can be used for commercial production. Regardless of the column,the column can be packed using a suitable media such as MabSelect™.

In certain aspects, the Protein A column can be equilibrated with asuitable buffer prior to sample loading. An example of a suitable bufferis a Tris/NaCl buffer, pH of about 6 to 8, and in certain embodimentsabout 7.2. A specific example of suitable conditions is 25 mM Tris, 100mM NaCl, pH 7.2. Following this equilibration, the sample can be loadedonto the column. Following the loading of the column, the column can bewashed one or multiple times using, e.g., the equilibrating buffer.Other washes including washes employing different buffers can be usedbefore eluting the column. For example, the column can be washed usingone or more column volumes of 20 mM citric acid/sodium citrate, 0.5 MNaCl at pH of about 6.0. This wash can optionally be followed by one ormore washes using the equilibrating buffer. The Protein A column canthen be eluted using an appropriate elution buffer. An example of asuitable elution buffer is an acetic acid/NaCl buffer, pH around 3.5.Suitable conditions are, e.g., 0.1 M acetic acid, pH 3.5. The eluate canbe monitored using techniques well known to those skilled in the art.For example, the absorbance at OD₂₈₀ can be followed. Column eluate canbe collected starting with an initial deflection of about 0.5 AU to areading of about 0.5 AU at the trailing edge of the elution peak. Theelution fraction(s) of interest can then be prepared for furtherprocessing. For example, the collected sample can be titrated to a pH ofabout 5.0 using Tris (e.g., 1.0 M) at a pH of about 10. Optionally, thistitrated sample can be filtered and further processed.

In certain embodiments, the HIC purification step can also be precededby an ion exchange chromatography step. The ion exchange purificationstep can occur before, after, or in place of an affinity chromatographystep. In certain embodiments, where a Protein A step precedes the ionexchange step, a Protein A eluate is purified using a cation exchangecolumn. In certain embodiments, the equilibrating buffer used in thecation exchange column is a buffer having a pH of about 5.0. An exampleof a suitable buffer is about 210 mM sodium acetate, pH 5.0. Followingequilibration, the column is loaded with sample prepared from HICpurification step above. The column is packed with a cation exchangemedia, such as CM Sepharose™ Fast Flow from GE Healthcare. The column isthen washed using the equilibrating buffer. The column is next subjectedto an elution step using a buffer having a greater ionic strength ascompared to the equilibrating or wash buffer. For example, a suitableelution buffer can be about 790 mM sodium acetate, pH 5.0. Theantibodies will be eluted and can be monitored using a UVspectrophotometer set at OD_(280nm). In a particular example, elutioncollection can be from upside 3 OD_(280nm) to downside 8 OD_(280nm). Itshould be understood that one skilled in the art may vary the conditionsand yet still be within the scope of the invention.

In certain embodiments where a Protein A step precedes an ion exchangestep, a Protein A eluate is purified using an anion exchange column. Anon-limiting example of a suitable column for this step is a 60 cmdiameter×30 cm long column whose bed volume is about 85 L. The column ispacked with an anion exchange media, such as Q Sepharose™ Fast Flow fromGE Healthcare. The column can be equilibrated using about seven columnvolumes of an appropriate buffer such as Tris/sodium chloride. Anexample of suitable conditions is 25 mM Tris, 50 mM sodium chloride atpH 8.0. A skilled artisan may vary the conditions but still be withinthe scope of the present invention. The column is loaded with thecollected sample from the HIC purification step outlined above. Inanother aspect, the column is loaded from the eluate collected duringcation exchange. Following the loading of the column, the column iswashed with the equilibration buffer (e.g., the Tris/sodium chloridebuffer). The flow-through comprising the antibodies can be monitoredusing a UV spectrophotometer at OD_(280nm). This anion exchange stepreduces process related impurities such as nucleic acids like DNA, andhost cell proteins. The separation occurs due to the fact that theantibodies of interest do not substantially interact with nor bind tothe solid phase of the column, e.g., to the Q Sepharose™, but manyimpurities do interact with and bind to the column's solid phase. Theanion exchange can be performed at about 12° C.

In certain embodiments, the cation exchange or anion exchange eluate,depending on which ion exchange step is employed, or employed first, isnext filtered using, e.g., a 16 inch Cuno™ delipid filter. Thisfiltration, using the delipid filter, can be followed by, e.g., a30-inch 0.45/0.2 μm Sartopore™ bi-layer filter cartridge. The ionexchange elution buffer can be used to flush the residual volumeremaining in the filters and prepared for ultrafiltration/diafiltration.

In order to accomplish the ultratfiltration/diafiltration step, thefiltration media is prepared in a suitable buffer, e.g., 20 mM sodiumphosphate, pH 7.0. A salt such as sodium chloride can be added toincrease the ionic strength, e.g., 100 mM sodium chloride. Thisultrafiltration/diafiltration step serves to concentrate the anti-IL-12,anti-TNFα, or anti-IL-18 antibodies, remove the sodium acetate andadjust the pH. Commercial filters are available to effectuate this step.For example, Millipore manufactures a 30 kD molecular weight cut-off(MWCO) cellulose ultrafilter membrane cassette. This filtrationprocedure can be conducted at or around room temperature.

In certain embodiments, the sample from the capture filtration stepabove is subjected to a second ion exchange separation step. In certainembodiments, this second ion exchange separation will involve separationbased on the opposite charge of the first ion exchange separation. Forexample, if an anion exchange step is employed after HIC purification,the second ion exchange chromatographic step may be a cation exchangestep. Conversely, if the HIC purification step was followed by a cationexchange step, that step would be followed by an anion exchange step. Incertain embodiments the first ion exchange eluate can be subjecteddirectly to the second ion exchange chromatographic step where the firstion exchange eluate is adjusted to the appropriate buffer conditions.Suitable anionic and cationic separation materials and conditions aredescribed above.

In certain embodiments, a mixed mode chromatography step will precedethe HIC chromatography step, thereby forming a mixed mode chromatographysample that can be exposed to the HIC media in the HIC chromatographystep. Examples of mixed mode medias include, but are not limited to:CaptoAdhere (GE Healthcare), PPA-HyperCel (Pall Life Sciences), andHEA-HyperCel (Pall Life Sciences). In certain embodiments, the mixedmode chromatography step is a CaptoAdhere chromatography step. Incertain embodiments, the mixed mode chromatography sample is furthersubject to a filtration step. Filters well known to those skilled in theart can be used in this embodiment. In one aspect, the filtration stepis a nanofiltration step. In certain embodiments, a depth filtrationstep follows a filtration step.

In certain embodiments of the invention, the wash and/or flow throughfractions from the hydrophobic chromatography step are subjected tofiltration for the removal of viral particles, including intact viruses,if present. A non-limiting example of a suitable filter is the UltiporDV50™ filter from Pall Corporation. Other viral filters can be used inthis filtration step and are well known to those skilled in the art. TheHIC eluate is passed through a pre-wetted filter of about 0.1 μm and a2×30-inch Ultipor DV50™ filter train at around 34 psig. In certainembodiments, following the filtration process, the filter is washedusing, e.g., the HIC wash buffer in order to remove any antibodiesretained in the filter housing. The filtrate can be stored in apre-sterilized container at around 12° C.

In a certain embodiments, the filtrate from the above is again subjectedto ultrafiltration/diafiltration. This step is important if apractitioner's end point is to use the antibody in a, e.g.,pharmaceutical formulation. This process, if employed, can facilitatethe concentration of antibody, removal of buffering salts previouslyused and replace it with a particular formulation buffer. In certainembodiments, continuous diafiltration with multiple volumes, e.g., twovolumes, of a formulation buffer is performed. A non-limiting example ofa suitable formulation buffer is 5 mM methionine, 2% mannitol, 0.5%sucrose, pH 5.9 buffer (no Tween). Upon completion of this diavolumeexchange the antibodies are concentrated. Once a predeterminedconcentration of antibody has been achieved, then a practitioner cancalculate the amount of 10% Tween that should be added to arrive at afinal Tween concentration of about 0.005% (v/v).

Certain embodiments of the present invention will include furtherpurification steps. Examples of additional purification procedures whichcan be performed prior to, during, or following the ion exchangechromatography method include ethanol precipitation, isoelectricfocusing, reverse phase HPLC, chromatography on silica, chromatographyon heparin Sepharose™ further anion exchange chromatography and/orfurther cation exchange chromatography, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography (e.g., usingprotein G, an antibody, a specific substrate, ligand or antigen as thecapture reagent).

In certain embodiments the unbound flow through and wash fractions canbe further fractionated and a combination of fractions providing atarget protein of interest purity can be pooled.

In certain embodiments the protein concentration can be adjusted toachieve a differential partitioning behavior between the protein ofinterest and the impurities such that the purity and/or yield can befurther improved.

In certain embodiments the loading can be performed at different proteinconcentrations during the loading operation to improve the productquality/yield of any particular purification step.

In certain embodiments the column temperature, can be independentlyvaried to improve the separation efficiency and/or yield of anyparticular purification step.

In certain embodiments, the loading and washing buffers can be differentor composed of mixtures of chemicals, while achieving similar“hydrophobic interaction” behavior such that the above novel separationcan be effected.

In certain embodiments, the loading and washing buffers can bedifferent, in terms of ionic strength or pH, while remainingsubstantially the same in function in terms of the washout of theprotein of interest achieved during the wash step.

In certain embodiments, the loading & washing steps can be controlled byin-line, at-line or off-line measurement of the impurity levels, eitherin the column effluent, or the collected pool or both, so as to achievethe protein of interest quality and/or yield.

In certain embodiments, the loading concentration can be dynamicallycontrolled by in-line or batch or continuous dilutions with buffers orother solutions to achieve the partitioning necessary to improve theseparation efficiency and/or yield.

In certain embodiments, additives such as amino acids, sugars, PEG, etccan be added to the load or wash steps to modulate the partitioningbehavior to achieve the separation efficiency and/or yield.

In certain embodiments, the separation can be performed on any type ofHIC media such as membranes, monoliths or depth filters that havehydrophobic interaction characteristics.

Mixed mode media can also be employed to enable this method, providedthe same functionality is achieved by appropriately adjusting the columnloading and/or washing conditions.

Methods of Assaying Sample Purity Assaying Aggregates

In certain embodiments, the levels of product-related substances, suchas aggregates, in either the initial sample or the flow through and/orwash fractions following the HIC steps of the present invention areanalyzed. For example, but not by way of limitation, the aggregatespresent in the Adalimumab process samples can be quantified according tothe following methods.

Aggregates may be measured using a size exclusion chromatographic (SEC)method whereby molecules are separated based on size and/or molecularweight such that larger molecules elute earlier from the column. Forexample, but not by way of limitation, a SEC columns useful for thedetection of aggregates include: TSK-gel G3000SWxL, 5 μm, 125 Å, 7.8×300mm column (Tosoh Bioscience), TSK-gel Super SW3000, 4 μm, 250 Å, 4.6×300mm column (Tosoh Bioscience), or Zorbax GF450 column (AgilentTechnologies). A further example of an SEC column for analysis ofmonomers and aggregates is the MAbPac™ SEC-1 (Thermo Scientific) columnwhich may be used under non-denaturing conditions, in both high- andlow-salt mobile phases, and with volatile eluents. In certainembodiments, the aforementioned columns are used along with an Agilentor a Shimazhu HPLC system. In a particular embodiment of SEC, aggregatesmay be quantified using a Zorbax GF450 column on an Agilent HPLC system.

In certain embodiments, sample injections are made under isocraticelution conditions using a mobile phase consisting of, for example, 100mM sodium sulfate and 100 mM sodium phosphate at pH 6.8, and detectedwith UV absorbance at 214 nm. In certain embodiments, the mobile phasewill consist of 1×PBS at pH 7.4, and elution profile detected with UVabsorbance at 280 nm.

The elution profile may be further analyzed using multiangle laserlight-scattering (MALS), to determine the apparent molecular weight ofeach peak, and allow identification as a dimer, tetramer, or other highmolecular weight species (FIG. 1). The elution profile may also befurther analyzed using sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). For example, the fraction is mixed witheither a non-reducing or reducing denaturing sample buffer, treated fortwo minutes at 98° C. in an Eppendorf Thermomixer Confort, then loadedin a 5% polyacrylamide tris-HCL gel alongside pre-stained broad rangemolecular weight markers. Electrophoresis is performed using a buffercomprising 0.3% (w/v) Tris, 1.44% (w/v) glycine and 0.1% SDS, pH 8.3.Separation is performed at a constant current of 100 V and at maximally50 mA for about 1 hour, followed by staining of the gel. In anotherembodiment, the aggregates may be analyzed and the molecular weightdetermined using high performance-size exclusion chromatography followedby native electrospray ionization time-of-flight mass spectrometry(ESI-TOF MS). Further methods for assaying levels of aggregates areprovided in the Examples below.

Assaying Host Cell Protein

The present invention also provides methods for determining the residuallevels of host cell protein (HCP) concentration in the initial sample orthe flow through and/or wash fractions following the HIC steps of thepresent invention. As described above, HCPs are desirably excluded fromthe final preparation. Exemplary HCPs include proteins originating fromthe source of the protein of interest production. Failure to identifyand sufficiently remove HCPs from the target protein of interest maylead to reduced efficacy and/or adverse subject reactions whenadministered in a therapeutic setting.

As used herein, the term “HCP ELISA” refers to an ELISA where theantibody used in the assay is specific to the HCPs produced from cells,e.g., CHO cells, used to generate the protein of interest. The antibodymay be produced according to conventional methods known to those ofskill in the art. For example, the antibody may be produced using HCPsobtained by sham production and purification runs, i.e., the same cellline used to produce the protein of interest is used, but the cell lineis not transfected with antibody DNA. In an exemplary embodiment, theantibody is produced using HPCs similar to those expressed in the cellexpression system of choice, i.e., the cell expression system used toproduce the protein of interest.

Generally, HCP ELISA comprises sandwiching a liquid sample comprisingHCPs between two layers of antibodies, i.e., a first antibody and asecond antibody. The sample is incubated during which time the HCPs inthe sample are captured by the first antibody, for example, but notlimited to goat anti-CHO, affinity purified (Cygnus). A labeled secondantibody, or blend of antibodies, specific to the HCPs produced from thecells used to generate the antibody, e.g., anti-CHO HCP Biotinylated, isadded, and binds to the HCPs within the sample. In certain embodiments,the first and second antibodies are polyclonal antibodies. In certainembodiments, the first and second antibodies are blends of polyclonalantibodies raised against HCPs. The amount of HCP contained in thesample is determined using the appropriate test based on the label ofthe second antibody.

HCP ELISA may be used for determining the level of HCPs in preparationor fraction, such as a wash fraction or a flow-through obtained usingthe process described above. The present invention also provides apreparation comprising a protein of interest, wherein the compositionhas no detectable level of HCPs as determined by an HCP Enzyme LinkedImmunosorbent Assay (“ELISA”). In one embodiment, the protein ofinterest is adalimumab.

Assaying Charge and Size Variants

In certain embodiments, the levels of product-related substances, suchas acidic species and other charge variants, in the chromatographicsamples produced using the techniques described herein are analyzed. Forexample, but not by way of limitation, the acidic species and othercharge variants present in the Adalimumab process samples can bequantified according to the following methods. Cation exchangechromatography was performed on a Dionex ProPac WCX-10, Analyticalcolumn 4 mm×250 mm (Dionex, CA). An Agilent 1200 HPLC system was used asthe HPLC. The mobile phases used were 10 mM Sodium Phosphate dibasic pH7.5 (Mobile phase A) and 10 mM Sodium Phosphate dibasic, 500 mM SodiumChloride pH 5.5 (Mobile phase B). A binary gradient (94% A, 6% B: 0-20min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B:28-34 min) was used with detection at 280 nm.

In certain embodiments, the levels of aggregates, monomer, and fragmentsin the chromatographic samples produced using the techniques describedherein are analyzed. In certain embodiments, the aggregates, monomer,and fragments are measured using a size exclusion chromatographic (SEC)method for each molecule. For example, but not by way of limitation, aTSK-gel G3000SWxL, 5 μm, 125 Å, 7.8×300 mm column (Tosoh Bioscience) canbe used in connection with certain embodiments, while a TSK-gel SuperSW3000, 4 μm, 250 Å, 4.6×300 mm column (Tosoh Bioscience) can be used inalternative embodiments. In certain embodiments, the aforementionedcolumns are used along with an Agilent or a Shimazhu HPLC system. Incertain embodiments, sample injections are made under isocratic elutionconditions using a mobile phase consisting of, for example, 100 mMsodium sulfate and 100 mM sodium phosphate at pH 6.8, and detected withUV absorbance at 214 nm. In certain embodiments, the mobile phase willconsist of 1×PBS at pH 7.4, and elution profile detected with UVabsorbance at 280 nm. In certain embodiments, quantification is based onthe relative area of detected peaks.

Antibody Generation

Antibodies to be purified by the methods of the present invention can begenerated by a variety of techniques, including immunization of ananimal with the antigen of interest followed by conventional monoclonalantibody methodologies e.g., the standard somatic cell hybridizationtechnique of Kohler and Milstein (1975) Nature 256: 495. Althoughsomatic cell hybridization procedures are preferred, in principle, othertechniques for producing monoclonal antibody can be employed e.g., viralor oncogenic transformation of B lymphocytes.

In certain embodiments, the animal system for preparing hybridomas isthe murine system. Hybridoma production is a well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

An antibody can be, in certain embodiments, a human, a chimeric, or ahumanized antibody. Humanized antibodies of the present disclosure canbe prepared based on the sequence of a non-human monoclonal antibodyprepared as described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the non-human hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,murine CDR regions can be inserted into a human framework using methodsknown in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen etal.).

Human monoclonal antibodies can be generated using transgenic ortranschromosomic mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice referred to herein as the HuMAb Mouse® (Medarex, Inc.), KMMouse® (Medarex, Inc.), and XenoMouse® (Amgen).

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseantibodies of the disclosure. For example, mice carrying both a humanheavy chain transchromosome and a human light chain tranchromosome,referred to as “TC mice” can be used; such mice are described inTomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727.Furthermore, cows carrying human heavy and light chain transchromosomeshave been described in the art (e.g., Kuroiwa et al. (2002) NatureBiotechnology 20:889-894 and PCT application No. WO 2002/092812) and canbe used to raise the antibodies of this disclosure.

In certain embodiments, the antibodies of this disclosure arerecombinant human antibodies, which can be isolated by screening of arecombinant combinatorial antibody library, e.g., a scFv phage displaylibrary, prepared using human VL and VH cDNAs prepared from mRNA derivedfrom human lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. In addition to commercially availablekits for generating phage display libraries (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612, the entireteachings of which are incorporated herein), examples of methods andreagents particularly amenable for use in generating and screeningantibody display libraries can be found in, e.g., Ladner et al. U.S.Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Doweret al. PCT Publication No. WO 91/17271; Winter et al. PCT PublicationNo. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679;Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCTPublication No. WO 92/01047; Garrard et al. PCT Publication No. WO92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982; the entire teachings of whichare incorporated herein.

Human monoclonal antibodies of this disclosure can also be preparedusing SCID mice into which human immune cells have been reconstitutedsuch that a human antibody response can be generated upon immunization.Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

The antibodies or antigen-binding portions thereof can be alteredwherein the constant region of the antibody is modified to reduce atleast one constant region-mediated biological effector function relativeto an unmodified antibody. To modify an antibody of the invention suchthat it exhibits reduced binding to the Fc receptor, the immunoglobulinconstant region segment of the antibody can be mutated at particularregions necessary for Fc receptor (FcR) interactions (see, e.g.,Canfield and Morrison (1991) J. Exp. Med. 173:1483-1491; and Lund et al.(1991) J. of Immunol. 147:2657-2662, the entire teachings of which areincorporated herein). Reduction in FcR binding ability of the antibodymay also reduce other effector functions which rely on FcR interactions,such as opsonization and phagocytosis and antigen-dependent cellularcytotoxicity.

Antibody Production

To express an antibody of the invention, DNAs encoding partial orfull-length light and heavy chains are inserted into one or moreexpression vector such that the genes are operatively linked totranscriptional and translational control sequences. (See, e.g., U.S.Pat. No. 6,914,128, the entire teaching of which is incorporated hereinby reference.) In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into a separate vector or, more typically, bothgenes are inserted into the same expression vector. The antibody genesare inserted into an expression vector by standard methods (e.g.,ligation of complementary restriction sites on the antibody genefragment and vector, or blunt end ligation if no restriction sites arepresent). Prior to insertion of the antibody or antibody-related lightor heavy chain sequences, the expression vector may already carryantibody constant region sequences. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, a recombinant expression vectorof the invention can carry one or more regulatory sequence that controlsthe expression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, e.g., in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), the entire teaching of which is incorporatedherein by reference. It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., the entireteachings of which are incorporated herein by reference.

In addition to the antibody chain genes and regulatory sequences, arecombinant expression vector of the invention may carry one or moreadditional sequences, such as a sequence that regulates replication ofthe vector in host cells (e.g., origins of replication) and/or aselectable marker gene. The selectable marker gene facilitates selectionof host cells into which the vector has been introduced (see e.g., U.S.Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., theentire teachings of which are incorporated herein by reference). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Suitable selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr− hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

An antibody of the invention can be prepared by recombinant expressionof immunoglobulin light and heavy chain genes in a host cell. To expressan antibody recombinantly, a host cell is transfected with one or morerecombinant expression vectors carrying DNA fragments encoding theimmunoglobulin light and heavy chains of the antibody such that thelight and heavy chains are expressed in the host cell and secreted intothe medium in which the host cells are cultured, from which medium theantibodies can be recovered. Standard recombinant DNA methodologies areused to obtain antibody heavy and light chain genes, incorporate thesegenes into recombinant expression vectors and introduce the vectors intohost cells, such as those described in Sambrook, Fritsch and Maniatis(eds), Molecular Cloning; A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.Nos. 4,816,397 & 6,914,128, the entire teachings of which areincorporated herein.

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is (are) transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it istheoretically possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, such as mammalian host cells, is suitable because sucheukaryotic cells, and in particular mammalian cells, are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss and Wood (1985) Immunology Today 6:12-13, theentire teaching of which is incorporated herein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed inDD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

Suitable mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr− CHO cells, described in Urlaub and Chasin, (1980) PNAS USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inKaufman and Sharp (1982) Mol. Biol. 159:601-621, the entire teachings ofwhich are incorporated herein by reference), NSO myeloma cells, COScells and SP2 cells. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or secretionof the antibody into the culture medium in which the host cells aregrown. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2), the entire teachings of which are incorporated herein byreference.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce an antibody may be cultured in a varietyof media. Commercially available media such as Ham's F10™ (Sigma),Minimal Essential Medium™ ((MEM), (Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium™ ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may beused as culture media for the host cells, the entire teachings of whichare incorporated herein by reference. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas gentamycin drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, in certain embodiments it may be desirable totransfect a host cell with DNA encoding either the light chain or theheavy chain (but not both) of an antibody of this invention. RecombinantDNA technology may also be used to remove some or all of the DNAencoding either or both of the light and heavy chains that is notnecessary for binding to the antigen to which the putative antibody ofinterest binds. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than the one to which theputative antibody of interest binds, depending on the specificity of theantibody of the invention, by crosslinking an antibody of the inventionto a second antibody by standard chemical crosslinking methods.

In a suitable system for recombinant expression of an antibody of theinvention, a recombinant expression vector encoding both the antibodyheavy chain and the antibody light chain is introduced into dhfr-CHOcells by calcium phosphate-mediated transfection. Within the recombinantexpression vector, the antibody heavy and light chain genes are eachoperatively linked to CMV enhancer/AdMLP promoter regulatory elements todrive high levels of transcription of the genes. The recombinantexpression vector also carries a DHFR gene, which allows for selectionof CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the antibody heavy andlight chains and intact antibody is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. In one aspect, if the antibody is produced intracellularly, as afirst step, the particulate debris, either host cells or lysed cells(e.g., resulting from homogenization), can be removed, e.g., bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems can be firstconcentrated using a commercially available protein concentrationfilter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit.

Prior to the process of the invention, procedures for purification ofantibodies from cell debris initially depend on the site of expressionof the antibody. Some antibodies can be secreted directly from the cellinto the surrounding growth media; others are made intracellularly. Forthe latter antibodies, the first step of a purification processtypically involves: lysis of the cell, which can be done by a variety ofmethods, including mechanical shear, osmotic shock, or enzymatictreatments. Such disruption releases the entire contents of the cellinto the homogenate, and in addition produces subcellular fragments thatare difficult to remove due to their small size. These are generallyremoved by differential centrifugation or by filtration. Where theantibody is secreted, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit. Where the antibody is secreted into the medium,the recombinant host cells can also be separated from the cell culturemedium, e.g., by tangential flow filtration. Antibodies can be furtherrecovered from the culture medium using the antibody purificationmethods of the invention.

Methods of Treatment Using the Low Impurity Compositions of theInvention

The low impurity compositions, for example, low aggregate compositions,of the invention may be used to treat any disorder in a subject forwhich the therapeutic protein of interest (e.g., an antibody or anantigen binding portion thereof) comprised in the composition isappropriate for treating.

A “disorder” is any condition that would benefit from treatment with theprotein of interest. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose thesubject to the disorder in question. In the case of an anti-TNFαantibody, or antigen binding portion thereof, such as adalimumab, atherapeutically effective amount of the low impurity composition may beadministered to treat a disorder in which TNFα activity is detrimental.

A disorder in which TNFα activity is detrimental includes a disorder inwhich inhibition of TNFα activity is expected to alleviate the symptomsand/or progression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of TNFα in a biologicalfluid of a subject suffering from the disorder (e.g., an increase in theconcentration of TNFα in serum, plasma, synovial fluid, etc. of thesubject), which can be detected, for example, using an anti-TNFαantibody.

TNFα has been implicated in the pathophysiology of a wide variety of aTNFα-related disorders including sepsis, infections, autoimmunediseases, transplant rejection and graft-versus-host disease (see e.g.,Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024to Moeller et al.; European Patent Publication No. 260 610 B1 byMoeller, A., et al. Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452;Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).Accordingly, the low impurity compositions of the invention may be usedto treat an autoimmune disease, such as rheumatoid arthritis, juvenileidiopathic arthritis, or psoriatic arthritis, an intestinal disorder,such as Crohn's disease or ulcerative colitis, a spondyloarthropathy,such as ankylosing spondylitis, or a skin disorder, such as psoriasis.

Disorders in which TNFα activity is detrimental are well known in theart and described in detail in U.S. Pat. No. 8,231,876 and U.S. Pat. No.6,090,382, the entire contents of each of which are expresslyincorporated herein by reference. In one embodiment, “a disorder inwhich TNFα activity is detrimental” includes sepsis (including septicshock, endotoxic shock, gram negative sepsis and toxic shock syndrome),autoimmune diseases (including rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis and gouty arthritis, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis, nephrotic syndrome,multisystem autoimmune diseases, lupus (including systemic lupus, lupusnephritis and lupus cerebritis), Crohn's disease and autoimmune hearingloss), infectious diseases (including malaria, meningitis, acquiredimmune deficiency syndrome (AIDS), influenza and cachexia secondary toinfection), allograft rejection and graft versus host disease,malignancy, pulmonary disorders (including adult respiratory distresssyndrome (ARDS), shock lung, chronic pulmonary inflammatory disease,pulmonary sarcoidosis, pulmonary fibrosis, silicosis, idiopathicinterstitial lung disease and chronic obstructive airway disorders(COPD), such as asthma), intestinal disorders (including inflammatorybowel disorders, idiopathic inflammatory bowel disease, Crohn's diseaseand Crohn's disease-related disorders (including fistulas in thebladder, vagina, and skin; bowel obstructions; abscesses; nutritionaldeficiencies; complications from corticosteroid use; inflammation of thejoints; erythem nodosum; pyoderma gangrenosum; lesions of the eye,Crohn's related arthralgias, fistulizing Crohn's indeterminant colitisand pouchitis), cardiac disorders (including ischemia of the heart,heart insufficiency, restenosis, congestive heart failure, coronaryartery disease, angina pectoris, myocardial infarction, cardiovasculartissue damage caused by cardiac arrest, cardiovascular tissue damagecaused by cardiac bypass, cardiogenic shock, and hypertension,atherosclerosis, cardiomyopathy, coronary artery spasm, coronary arterydisease, valvular disease, arrhythmias, and cardiomyopathies),spondyloarthropathies (including ankylosing spondylitis, psoriaticarthritis/spondylitis, enteropathic arthritis, reactive arthritis orReiter's syndrome, and undifferentiated spondyloarthropathies),metabolic disorders (including obesity and diabetes, including type 1diabetes mellitus, type 2 diabetes mellitus, diabetic neuropathy,peripheral neuropathy, diabetic retinopathy, diabetic ulcerations,retinopathy ulcerations and diabetic macrovasculopathy), anemia, pain(including acute and chronic pains, such as neuropathic pain andpost-operative pain, chronic lower back pain, cluster headaches, herpesneuralgia, phantom limb pain, central pain, dental pain,opioid-resistant pain, visceral pain, surgical pain, bone injury pain,pain during labor and delivery, pain resulting from burns, includingsunburn, post partum pain, migraine, angina pain, and genitourinarytract-related pain including cystitis), hepatic disorders (includinghepatitis, alcoholic hepatitis, viral hepatitis, alcoholic cirrhosis, a1antitypsin deficiency, autoimmune cirrhosis, cryptogenic cirrhosis,fulminant hepatitis, hepatitis B and C, and steatohepatitis, cysticfibrosis, primary biliary cirrhosis, sclerosing cholangitis and biliaryobstruction), skin and nail disorders (including psoriasis (includingchronic plaque psoriasis, guttate psoriasis, inverse psoriasis, pustularpsoriasis and other psoriasis disorders), pemphigus vulgaris,scleroderma, atopic dermatitis (eczema), sarcoidosis, erythema nodosum,hidradenitis suppurative, lichen planus, Sweet's syndrome, sclerodermaand vitiligo), vasculitides (including Behcet's disease), and otherdisorders, such as juvenile rheumatoid arthritis (JRA), endometriosis,prostatitis, choroidal neovascularization, sciatica, Sjogren's syndrome,uveitis, wet macular degeneration, osteoporosis and osteoarthritis.

As used herein, the term “subject” is intended to include livingorganisms, e.g., prokaryotes and eukaryotes. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats, and transgenic non-human animals. In specificembodiments of the invention, the subject is a human.

As used herein, the term “treatment” or “treat” refers to boththerapeutic treatment and prophylactic or preventative measures. Thosein need of treatment include those already with the disorder, as well asthose in which the disorder is to be prevented.

In one embodiment, the invention provides a method of administering alow impurity composition comprising an anti-TNFα antibody, or antigenbinding portion thereof, to a subject such that TNFα activity isinhibited or a disorder in which TNFα activity is detrimental istreated. In one embodiment, the TNFα is human TNFα and the subject is ahuman subject. In one embodiment, the anti-TNFα antibody is adalimumab,also referred to as HUMIRA®.

The low impurity compositions can be administered by a variety ofmethods known in the art. Exemplary routes/modes of administrationinclude subcutaneous injection, intravenous injection or infusion. Incertain aspects, a low impurity compositions may be orally administered.As will be appreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. In certainembodiments it is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit comprising a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a low impurity composition of theinvention is 0.01-20 mg/kg, or 1-10 mg/kg, or 0.3-1 mg/kg. With respectto low impurity compositions comprising an anti-TNFα antibody, orantigen-binding portion thereof, such as adalimumab, an exemplary doseis 40 mg every other week. In some embodiments, in particular fortreatment of ulcerative colitis or Crohn's disease, an exemplary doseincludes an initial dose (Day 1) of 160 mg (e.g., four 40 mg injectionsin one day or two 40 mg injections per day for two consecutive days), asecond dose two weeks later of 80 mg, and a maintenance dose of 40 mgevery other week beginning two weeks later. Alternatively, for psoriasisfor example, a dosage can include an 80 mg initial dose followed by 40mg every other week starting one week after the initial dose.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

Pharmaceutical Formulations Containing the Low Impurity Compositions ofthe Invention

The present invention further provides preparations and formulationscomprising low impurity compositions, for example, low aggregatecompositions, of the invention. It should be understood that any of theproteins of interest, such as antibodies and antibody fragmentsdescribed herein, including proteins of interest having any one or moreof the structural and functional features described in detail throughoutthe application, may be formulated or prepared as described below. Whenvarious formulations are described in this section as including aprotein of interest, such as an antibody, it is understood that such aprotein of interest may be a protein having any one or more of thecharacteristics of the proteins of interest described herein. In oneembodiment, the antibody is an anti-TNFα antibody, or antigen-bindingportion thereof.

In certain embodiments, the low impurity compositions, for example, lowaggregate compositions, of the invention may be formulated with apharmaceutically acceptable carrier as pharmaceutical (therapeutic)compositions, and may be administered by a variety of methods known inthe art. As will be appreciated by the skilled artisan, the route and/ormode of administration will vary depending upon the desired results. Theterm “pharmaceutically acceptable carrier” means one or more non-toxicmaterials that do not interfere with the effectiveness of the biologicalactivity of the active ingredients. Such preparations may routinelycontain salts, buffering agents, preservatives, compatible carriers, andoptionally other therapeutic agents. Such pharmaceutically acceptablepreparations may also routinely contain compatible solid or liquidfillers, diluents or encapsulating substances which are suitable foradministration into a human. The term “carrier” denotes an organic orinorganic ingredient, natural or synthetic, with which the activeingredient is combined to facilitate the application. The components ofthe pharmaceutical compositions also are capable of being co-mingledwith the proteins of interest (e.g., antibodies) of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

The low impurity compositions, for example, low aggregate compositions,of the invention are present in a form known in the art and acceptablefor therapeutic uses. In one embodiment, a formulation of the lowimpurity compositions, for example, low aggregate compositions, of theinvention is a liquid formulation. In another embodiment, a formulationof the low impurity compositions, for example, low aggregatecompositions, of the invention is a lyophilized formulation. In afurther embodiment, a formulation of the low impurity compositions, forexample, low aggregate compositions, of the invention is a reconstitutedliquid formulation. In one embodiment, a formulation of the low impuritycompositions, for example, low aggregate compositions, of the inventionis a stable liquid formulation. In one embodiment, a liquid formulationof the low impurity compositions, for example, low aggregatecompositions, of the invention is an aqueous formulation. In anotherembodiment, the liquid formulation is non-aqueous. In a specificembodiment, a liquid formulation of the low impurity compositions, forexample, low aggregate compositions, of the invention is an aqueousformulation wherein the aqueous carrier is distilled water.

The formulations of the low impurity compositions, for example, lowaggregate compositions, of the invention comprise a protein of interest(e.g., an antibody) in a concentration resulting in a w/v appropriatefor a desired dose. The protein of interest may be present in theformulation at a concentration of about 1 mg/ml to about 500 mg/ml,e.g., at a concentration of at least 1 mg/ml, at least 5 mg/ml, at least10 mg/ml, at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, atleast 30 mg/ml, at least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml,at least 50 mg/ml, at least 55 mg/ml, at least 60 mg/ml, at least 65mg/ml, at least 70 mg/ml, at least 75 mg/ml, at least 80 mg/ml, at least85 mg/ml, at least 90 mg/ml, at least 95 mg/ml, at least 100 mg/ml, atleast 105 mg/ml, at least 110 mg/ml, at least 115 mg/ml, at least 120mg/ml, at least 125 mg/ml, at least 130 mg/ml, at least 135 mg/ml, atleast 140 mg/ml, at least 150 mg/ml, at least 200 mg/ml, at least 250mg/ml, or at least 300 mg/ml.

In a specific embodiment, a formulation of the low impuritycompositions, for example, low aggregate compositions, of the inventioncomprises at least about 100 mg/ml, at least about 125 mg/ml, at least130 mg/ml, or at least about 150 mg/ml of protein of interest (e.g., anantibody) of the invention.

In one embodiment, the concentration of protein of interest (e.g.,antibody), which is included in the formulation of the invention, isbetween about 1 mg/ml and about 25 mg/ml, between about 1 mg/ml andabout 200 mg/ml, between about 25 mg/ml and about 200 mg/ml, betweenabout 50 mg/ml and about 200 mg/ml, between about 75 mg/ml and about 200mg/ml, between about 100 mg/ml and about 200 mg/ml, between about 125mg/ml and about 200 mg/ml, between about 150 mg/ml and about 200 mg/ml,between about 25 mg/ml and about 150 mg/ml, between about 50 mg/ml andabout 150 mg/ml, between about 75 mg/ml and about 150 mg/ml, betweenabout 100 mg/ml and about 150 mg/ml, between about 125 mg/ml and about150 mg/ml, between about 25 mg/ml and about 125 mg/ml, between about 50mg/ml and about 125 mg/ml, between about 75 mg/ml and about 125 mg/ml,between about 100 mg/ml and about 125 mg/ml, between about 25 mg/ml andabout 100 mg/ml, between about 50 mg/ml and about 100 mg/ml, betweenabout 75 mg/ml and about 100 mg/ml, between about 25 mg/ml and about 75mg/ml, between about 50 mg/ml and about 75 mg/ml, or between about 25mg/ml and about 50 mg/ml.

In a specific embodiment, a formulation of the low impuritycompositions, for example, low aggregate compositions, of the inventioncomprises between about 90 mg/ml and about 110 mg/ml or between about100 mg/ml and about 210 mg/ml of a protein of interest (e.g., anantibody).

The formulations of the low impurity compositions, for example, lowaggregate compositions, of the invention comprising a protein ofinterest (e.g., an antibody) may further comprise one or more activecompounds as necessary for the particular indication being treated,typically those with complementary activities that do not adverselyaffect each other. Such additional active compounds are suitably presentin combination in amounts that are effective for the purpose intended.

The formulations of the low impurity compositions, for example, lowaggregate compositions, of the invention may be prepared for storage bymixing the protein of interest (e.g., antibody) having the desireddegree of purity with optional physiologically acceptable carriers,excipients or stabilizers, including, but not limited to bufferingagents, saccharides, salts, surfactants, solubilizers, polyols,diluents, binders, stabilizers, salts, lipophilic solvents, amino acids,chelators, preservatives, or the like (Goodman and Gilman's ThePharmacological Basis of Therapeutics, 12^(th) edition, L. Brunton, etal. and Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.(1999)), in the form of lyophilized formulations or aqueous solutions ata desired final concentration. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as histidine, phosphate, citrate,glycine, acetate and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including trehalose, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g., Zn-protein complexes); and/or non-ionic surfactantssuch as TWEEN, polysorbate 80, PLURONICS™ or polyethylene glycol (PEG).

The buffering agent may be histidine, citrate, phosphate, glycine, oracetate. The saccharide excipient may be trehalose, sucrose, mannitol,maltose or raffinose. The surfactant may be polysorbate 20, polysorbate40, polysorbate 80, or Pluronic F68. The salt may be NaCl, KCl, MgCl₂,or CaCl₂

The formulations of the low impurity compositions, for example, lowaggregate compositions, of the invention may include a buffering or pHadjusting agent to provide improved pH control. A formulation of theinvention may have a pH of between about 3.0 and about 9.0, betweenabout 4.0 and about 8.0, between about 5.0 and about 8.0, between about5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5and about 8.0, between about 5.5 and about 7.0, or between about 5.5 andabout 6.5. In a further embodiment, a formulation of the invention has apH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1,about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4,about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about7.5, about 8.0, about 8.5, or about 9.0. In a specific embodiment, aformulation of the invention has a pH of about 6.0. One of skill in theart understands that the pH of a formulation generally should not beequal to the isoelectric point of the particular protein of interest(e.g., antibody) to be used in the formulation.

Typically, the buffering agent is a salt prepared from an organic orinorganic acid or base. Representative buffering agents include, but arenot limited to, organic acid salts such as salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride,or phosphate buffers. In addition, amino acid components can alsofunction in a buffering capacity. Representative amino acid componentswhich may be utilized in the formulations of the invention as bufferingagents include, but are not limited to, glycine and histidine. Incertain embodiments, the buffering agent is chosen from histidine,citrate, phosphate, glycine, and acetate. In a specific embodiment, thebuffering agent is histidine. In another specific embodiment, thebuffering agent is citrate. In yet another specific embodiment, thebuffering agent is glycine. The purity of the buffering agent should beat least 98%, or at least 99%, or at least 99.5%. As used herein, theterm “purity” in the context of histidine and glycine refers to chemicalpurity of histidine or glycine as understood in the art, e.g., asdescribed in The Merck Index, 13^(th) ed., O'Neil et al. ed. (Merck &Co., 2001).

Buffering agents are typically used at concentrations between about 1 mMand about 200 mM or any range or value therein, depending on the desiredionic strength and the buffering capacity required. The usualconcentrations of conventional buffering agents employed in parenteralformulations can be found in: Pharmaceutical Dosage Form: ParenteralMedications, Volume 1, 2^(nd) Edition, Chapter 5, p. 194, De Luca andBoylan, “Formulation of Small Volume Parenterals”, Table 5: Commonlyused additives in Parenteral Products. In one embodiment, the bufferingagent is at a concentration of about 1 mM, or of about 5 mM, or of about10 mM, or of about 15 mM, or of about 20 mM, or of about 25 mM, or ofabout 30 mM, or of about 35 mM, or of about 40 mM, or of about 45 mM, orof about 50 mM, or of about 60 mM, or of about 70 mM, or of about 80 mM,or of about 90 mM, or of about 100 mM. In one embodiment, the bufferingagent is at a concentration of 1 mM, or of 5 mM, or of 10 mM, or of 15mM, or of 20 mM, or of 25 mM, or of 30 mM, or of 35 mM, or of 40 mM, orof 45 mM, or of 50 mM, or of 60 mM, or of 70 mM, or of 80 mM, or of 90mM, or of 100 mM. In a specific embodiment, the buffering agent is at aconcentration of between about 5 mM and about 50 mM. In another specificembodiment, the buffering agent is at a concentration of between 5 mMand 20 mM.

In certain embodiments, the formulation of the low impuritycompositions, for example, low aggregate compositions, of the inventioncomprises histidine as a buffering agent. In one embodiment thehistidine is present in the formulation of the invention at aconcentration of at least about 1 mM, at least about 5 mM, at leastabout 10 mM, at least about 20 mM, at least about 30 mM, at least about40 mM, at least about 50 mM, at least about 75 mM, at least about 100mM, at least about 150 mM, or at least about 200 mM histidine. Inanother embodiment, a formulation of the invention comprises betweenabout 1 mM and about 200 mM, between about 1 mM and about 150 mM,between about 1 mM and about 100 mM, between about 1 mM and about 75 mM,between about 10 mM and about 200 mM, between about 10 mM and about 150mM, between about 10 mM and about 100 mM, between about 10 mM and about75 mM, between about 10 mM and about 50 mM, between about 10 mM andabout 40 mM, between about 10 mM and about 30 mM, between about 20 mMand about 75 mM, between about 20 mM and about 50 mM, between about 20mM and about 40 mM, or between about 20 mM and about 30 mM histidine. Ina further embodiment, the formulation comprises about 1 mM, about 5 mM,about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM,about 90 mM, about 100 mM, about 150 mM, or about 200 mM histidine. In aspecific embodiment, a formulation may comprise about 10 mM, about 25mM, or no histidine.

The formulations of the low impurity compositions, for example, lowaggregate compositions, of the invention may comprise a carbohydrateexcipient. Carbohydrate excipients can act, e.g., as viscosity enhancingagents, stabilizers, bulking agents, solubilizing agents, and/or thelike. Carbohydrate excipients are generally present at between about 1%to about 99% by weight or volume, e.g., between about 0.1% to about 20%,between about 0.1% to about 15%, between about 0.1% to about 5%, betweenabout 1% to about 20%, between about 5% to about 15%, between about 8%to about 10%, between about 10% and about 15%, between about 15% andabout 20%, between 0.1% to 20%, between 5% to 15%, between 8% to 10%,between 10% and 15%, between 15% and 20%, between about 0.1% to about5%, between about 5% to about 10%, or between about 15% to about 20%. Instill other specific embodiments, the carbohydrate excipient is presentat 1%, or at 1.5%, or at 2%, or at 2.5%, or at 3%, or at 4%, or at 5%,or at 10%, or at 15%, or at 20%.

Carbohydrate excipients suitable for use in the formulations of theinvention include, but are not limited to, monosaccharides such asfructose, maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.In one embodiment, the carbohydrate excipients for use in the presentinvention are chosen from, sucrose, trehalose, lactose, mannitol, andraffinose. In a specific embodiment, the carbohydrate excipient istrehalose. In another specific embodiment, the carbohydrate excipient ismannitol. In yet another specific embodiment, the carbohydrate excipientis sucrose. In still another specific embodiment, the carbohydrateexcipient is raffinose. The purity of the carbohydrate excipient shouldbe at least 98%, or at least 99%, or at least 99.5%.

In a specific embodiment, the formulations of the low impuritycompositions, for example, low aggregate compositions, of the inventionmay comprise trehalose. In one embodiment, a formulation of theinvention comprises at least about 1%, at least about 2%, at least about4%, at least about 8%, at least about 20%, at least about 30%, or atleast about 40% trehalose. In another embodiment, a formulation of theinvention comprises between about 1% and about 40%, between about 1% andabout 30%, between about 1% and about 20%, between about 2% and about40%, between about 2% and about 30%, between about 2% and about 20%,between about 4% and about 40%, between about 4% and about 30%, orbetween about 4% and about 20% trehalose. In a further embodiment, aformulation of the invention comprises about 1%, about 2%, about 4%,about 6%, about 8%, about 15%, about 20%, about 30%, or about 40%trehalose. In a specific embodiment, a formulation of the inventioncomprises about 4%, about 6% or about 15% trehalose.

In certain embodiments, a formulation of the low impurity compositions,for example, low aggregate compositions, of the invention comprises anexcipient. In a specific embodiment, a formulation of the inventioncomprises at least one excipient chosen from: sugar, salt, surfactant,amino acid, polyol, chelating agent, emulsifier and preservative. In oneembodiment, a formulation of the invention comprises a salt, e.g., asalt selected from: NaCl, KCl, CaCl₂, and MgCl₂. In a specificembodiment, the formulation comprises NaCl.

A formulation of the low impurity compositions, for example, lowaggregate compositions, of the invention may comprise at least about 10mM, at least about 25 mM, at least about 50 mM, at least about 75 mM, atleast about 80 mM, at least about 100 mM, at least about 125 mM, atleast about 150 mM, at least about 175 mM, at least about 200 mM, or atleast about 300 mM sodium chloride (NaCl). In a further embodiment, theformulation may comprise between about 10 mM and about 300 mM, betweenabout 10 mM and about 200 mM, between about 10 mM and about 175 mM,between about 10 mM and about 150 mM, between about 25 mM and about 300mM, between about 25 mM and about 200 mM, between about 25 mM and about175 mM, between about 25 mM and about 150 mM, between about 50 mM andabout 300 mM, between about 50 mM and about 200 mM, between about 50 mMand about 175 mM, between about 50 mM and about 150 mM, between about 75mM and about 300 mM, between about 75 mM and about 200 mM, between about75 mM and about 175 mM, between about 75 mM and about 150 mM, betweenabout 100 mM and about 300 mM, between about 100 mM and about 200 mM,between about 100 mM and about 175 mM, or between about 100 mM and about150 mM sodium chloride. In a further embodiment, the formulation maycomprise about 10 mM, about 25 mM, about 50 mM, about 75 mM, about 80mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200mM, or about 300 mM sodium chloride.

A formulation of the low impurity compositions, for example, lowaggregate compositions, of the invention may also comprise an aminoacid, e.g., lysine, arginine, glycine, histidine or an amino acid salt.The formulation may comprise at least about 1 mM, at least about 10 mM,at least about 25 mM, at least about 50 mM, at least about 100 mM, atleast about 150 mM, at least about 200 mM, at least about 250 mM, atleast about 300 mM, at least about 350 mM, or at least about 400 mM ofan amino acid. In another embodiment, the formulation may comprisebetween about 1 mM and about 100 mM, between about 10 mM and about 150mM, between about 25 mM and about 250 mM, between about 25 mM and about300 mM, between about 25 mM and about 350 mM, between about 25 mM andabout 400 mM, between about 50 mM and about 250 mM, between about 50 mMand about 300 mM, between about 50 mM and about 350 mM, between about 50mM and about 400 mM, between about 100 mM and about 250 mM, betweenabout 100 mM and about 300 mM, between about 100 mM and about 400 mM,between about 150 mM and about 250 mM, between about 150 mM and about300 mM, or between about 150 mM and about 400 mM of an amino acid. In afurther embodiment, a formulation of the invention comprises about 1 mM,1.6 mM, 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM,about 250 mM, about 300 mM, about 350 mM, or about 400 mM of an aminoacid.

The formulations of the low impurity compositions, for example, lowaggregate compositions, of the invention may further comprise asurfactant. The term “surfactant” as used herein refers to organicsubstances having amphipathic structures; namely, they are composed ofgroups of opposing solubility tendencies, typically an oil-solublehydrocarbon chain and a water-soluble ionic group. Surfactants can beclassified, depending on the charge of the surface-active moiety, intoanionic, cationic, and nonionic surfactants. Surfactants are often usedas wetting, emulsifying, solubilizing, and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.Pharmaceutically acceptable surfactants like polysorbates (e.g.,polysorbates 20 or 80); polyoxamers (e.g., poloxamer 188); Triton;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUA™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.), canoptionally be added to the formulations of the invention to reduceaggregation. In one embodiment, a formulation of the invention comprisesPolysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80.Surfactants are particularly useful if a pump or plastic container isused to administer the formulation. The presence of a pharmaceuticallyacceptable surfactant mitigates the propensity for the protein toaggregate. The formulations may comprise a polysorbate which is at aconcentration ranging from between about 0.001% to about 1%, or about0.001% to about 0.1%, or about 0.01% to about 0.1%. In other specificembodiments, the formulations of the invention comprise a polysorbatewhich is at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%,or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or0.015%, or 0.02%.

The formulations of the low impurity compositions, for example, lowaggregate compositions, of the invention may optionally further compriseother common excipients and/or additives including, but not limited to,diluents, binders, stabilizers, lipophilic solvents, preservatives,adjuvants, or the like. Pharmaceutically acceptable excipients and/oradditives may be used in the formulations of the invention. Commonlyused excipients/additives, such as pharmaceutically acceptable chelators(for example, but not limited to, EDTA, DTPA or EGTA) can optionally beadded to the formulations of the invention to reduce aggregation. Theseadditives are particularly useful if a pump or plastic container is usedto administer the formulation.

Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (for example, but notlimited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl andthe like), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal, or mixtures thereof can optionally beadded to the formulations of the invention at any suitable concentrationsuch as between about 0.001% to about 5%, or any range or value therein.The concentration of preservative used in the formulations of theinvention is a concentration sufficient to yield a microbial effect.Such concentrations are dependent on the preservative selected and arereadily determined by the skilled artisan.

Other contemplated excipients/additives, which may be utilized in theformulations of the invention include, for example, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,lipids such as phospholipids or fatty acids, steroids such ascholesterol, protein excipients such as serum albumin (human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,salt-forming counterions such as sodium and the like. These andadditional known pharmaceutical excipients and/or additives suitable foruse in the formulations of the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 21^(st) ed.,Lippincott Williams & Wilkins, (2005), and in the “Physician's DeskReference”, 60^(th) ed., Medical Economics, Montvale, N.J. (2005).Pharmaceutically acceptable carriers can be routinely selected that aresuitable for the mode of administration, solubility and/or stability ofprotein of interest (e.g., an antibody), as well known those in the artor as described herein.

In one embodiment, the low impurity compositions, for example, lowaggregate compositions, of the invention are formulated with the same orsimilar excipients and buffers as are present in the commercialadalimumab (HUMIRA®) formulation, as described in the “Highlights ofPrescribing Information” for HUMIRA® (adalimumab) Injection (RevisedJanuary 2008) the contents of which are hereby incorporated herein byreference. For example, each prefilled syringe of HUMIRA®, which isadministered subcutaneously, delivers 0.8 mL (40 mg) of drug product tothe subject. Each 0.8 mL of HUMIRA® contains 40 mg adalimumab, 4.93 mgsodium chloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22 mgdibasic sodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 mgcitric acid monohydrate, 9.6 mg mannitol, 0.8 mg polysorbate 80, andwater for Injection, USP. Sodium hydroxide is added as necessary toadjust pH.

It will be understood by one skilled in the art that the formulations ofthe low impurity compositions, for example, low aggregate compositions,of the invention may be isotonic with human blood, wherein theformulations of the invention have essentially the same osmotic pressureas human blood. Such isotonic formulations will generally have anosmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity canbe measured by, for example, using a vapor pressure or ice-freezing typeosmometer. Tonicity of a formulation is adjusted by the use of tonicitymodifiers. “Tonicity modifiers” are those pharmaceutically acceptableinert substances that can be added to the formulation to provide anisotonicity of the formulation. Tonicity modifiers suitable for thisinvention include, but are not limited to, saccharides, salts and aminoacids.

In certain embodiments, the formulations of the low impuritycompositions, for example, low aggregate compositions, of the inventionhave an osmotic pressure from about 100 mOSm to about 1200 mOSm, or fromabout 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about 800mOSm, or from about 200 mOSm to about 600 mOSm, or from about 250 mOSmto about 500 mOSm, or from about 250 mOSm to about 400 mOSm, or fromabout 250 mOSm to about 350 mOSm.

The concentration of any one component or any combination of variouscomponents, of the formulations of the low impurity compositions, forexample, low aggregate compositions, of the invention is adjusted toachieve the desired tonicity of the final formulation. For example, theratio of the carbohydrate excipient to protein of interest (e.g.,antibody) may be adjusted according to methods known in the art (e.g.,U.S. Pat. No. 6,685,940). In certain embodiments, the molar ratio of thecarbohydrate excipient to protein of interest (e.g., antibody) may befrom about 100 moles to about 1000 moles of carbohydrate excipient toabout 1 mole of protein of interest, or from about 200 moles to about6000 moles of carbohydrate excipient to about 1 mole of protein ofinterest, or from about 100 moles to about 510 moles of carbohydrateexcipient to about 1 mole of protein of interest, or from about 100moles to about 600 moles of carbohydrate excipient to about 1 mole ofprotein of interest.

The desired isotonicity of the final formulation may also be achieved byadjusting the salt concentration of the formulations. Pharmaceuticallyacceptable salts and those suitable for this invention as tonicitymodifiers include, but are not limited to, sodium chloride, sodiumsuccinate, sodium sulfate, potassium chloride, magnesium chloride,magnesium sulfate, and calcium chloride. In specific embodiments,formulations of the invention comprise NaCl, MgCl₂, and/or CaCl₂. In oneembodiment, concentration of NaCl is between about 75 mM and about 150mM. In another embodiment, concentration of MgCl₂ is between about 1 mMand about 100 mM. Pharmaceutically acceptable amino acids includingthose suitable for this invention as tonicity modifiers include, but arenot limited to, proline, alanine, L-arginine, asparagine, L-asparticacid, glycine, serine, lysine, and histidine.

In one embodiment the formulations of the low impurity compositions, forexample, low aggregate compositions, of the invention are pyrogen-freeformulations which are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released only when the microorganisms are brokendown or die. Pyrogenic substances also include fever-inducing,thermostable substances (glycoproteins) from the outer membrane ofbacteria and other microorganisms. Both of these substances can causefever, hypotension and shock if administered to humans. Due to thepotential harmful effects, even low amounts of endotoxins must beremoved from intravenously administered pharmaceutical drug solutions.The Food & Drug Administration (“FDA”) has set an upper limit of 5endotoxin units (EU) per dose per kilogram body weight in a single onehour period for intravenous drug applications (The United StatesPharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). Whentherapeutic proteins are administered in amounts of several hundred orthousand milligrams per kilogram body weight, as can be the case withproteins of interest (e.g., antibodies), even trace amounts of harmfuland dangerous endotoxin must be removed. In certain specificembodiments, the endotoxin and pyrogen levels in the composition areless then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or lessthen 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.

When used for in vivo administration, the formulations of the lowimpurity compositions, for example, low aggregate compositions, of theinvention should be sterile. The formulations of the invention may besterilized by various sterilization methods, including sterilefiltration, radiation, etc. In one embodiment, the protein of interest(e.g., antibody) formulation is filter-sterilized with a presterilized0.22-micron filter. Sterile compositions for injection can be formulatedaccording to conventional pharmaceutical practice as described in“Remington: The Science & Practice of Pharmacy”, 21^(st) ed., LippincottWilliams & Wilkins, (2005). Formulations comprising proteins of interest(e.g., antibodies), such as those disclosed herein, ordinarily will bestored in lyophilized form or in solution. It is contemplated thatsterile compositions comprising proteins of interest (e.g., antibodies)are placed into a container having a sterile access port, for example,an intravenous solution bag or vial having an adapter that allowsretrieval of the formulation, such as a stopper pierceable by ahypodermic injection needle. In one embodiment, a composition of theinvention is provided as a pre-filled syringe.

In one embodiment, a formulation of the low impurity compositions, forexample, low aggregate compositions, of the invention is a lyophilizedformulation. The term “lyophilized” or “freeze-dried” includes a stateof a substance that has been subjected to a drying procedure such aslyophilization, where at least 50% of moisture has been removed.

The phrase “bulking agent” includes a compound that is pharmaceuticallyacceptable and that adds bulk to a lyo cake. Bulking agents known to theart include, for example, carbohydrates, including simple sugars such asdextrose, ribose, fructose and the like, alcohol sugars such asmannitol, inositol and sorbitol, disaccharides including trehalose,sucrose and lactose, naturally occurring polymers such as starch,dextrans, chitosan, hyaluronate, proteins (e.g., gelatin and serumalbumin), glycogen, and synthetic monomers and polymers.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest (such as an antibody of the invention), significantly preventsor reduces chemical and/or physical instability of the protein uponlyophilization and subsequent storage. Lyoprotectants include, but arenot limited to, sugars and their corresponding sugar alcohols; an aminoacid such as monosodium glutamate or histidine; a methylamine such asbetaine; a lyotropic salt such as magnesium sulfate; a polyol such astrihydric or higher molecular weight sugar alcohols, e.g., glycerin,dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, andmannitol; propylene glycol; polyethylene glycol; PLURONICS™; andcombinations thereof. Additional examples of lyoprotectants include, butare not limited to, glycerin and gelatin, and the sugars mellibiose,melezitose, raffinose, mannotriose and stachyose. Examples of reducingsugars include, but are not limited to, glucose, maltose, lactose,maltulose, iso-maltulose and lactulose. Examples of non-reducing sugarsinclude, but are not limited to, non-reducing glycosides of polyhydroxycompounds selected from sugar alcohols and other straight chainpolyalcohols. Examples of sugar alcohols include, but are not limitedto, monoglycosides, compounds obtained by reduction of disaccharidessuch as lactose, maltose, lactulose and maltulose. The glycosidic sidegroup can be either glucosidic or galactosidic. Additional examples ofsugar alcohols include, but are not limited to, glucitol, maltitol,lactitol and iso-maltulose. In specific embodiments, trehalose orsucrose is used as a lyoprotectant.

The lyoprotectant is added to the pre-lyophilized formulation in a“lyoprotecting amount” which means that, following lyophilization of theprotein in the presence of the lyoprotecting amount of thelyoprotectant, the protein essentially retains its physical and chemicalstability and integrity upon lyophilization and storage.

In one embodiment, the molar ratio of a lyoprotectant (e.g., trehalose)and protein of interest (e.g., antibody) molecules of a formulation ofthe invention is at least about 10, at least about 50, at least about100, at least about 200, or at least about 300. In another embodiment,the molar ratio of a lyoprotectant (e.g., trehalose) and protein ofinterest molecules of a formulation of the invention is about 1, isabout 2, is about 5, is about 10, about 50, about 100, about 200, orabout 300.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized protein of interest (e.g., antibody)formulation in a diluent such that the protein of interest is dispersedin the reconstituted formulation. The reconstituted formulation issuitable for administration (e.g., parenteral administration) to apatient to be treated with the protein of interest and, in certainembodiments of the invention, may be one which is suitable forintravenous administration.

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, such as aformulation reconstituted after lyophilization. In some embodiments,diluents include, but are not limited to, sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution. In an alternative embodiment, diluents can includeaqueous solutions of salts and/or buffers.

In certain embodiments, a formulation of the low impurity compositions,for example, low aggregate compositions, of the invention is alyophilized formulation comprising a protein of interest (e.g.,antibody) of the invention, wherein at least about 90%, at least about95%, at least about 97%, at least about 98%, or at least about 99% ofsaid protein of interest may be recovered from a vial upon shaking saidvial for 4 hours at a speed of 400 shakes per minute wherein the vial isfilled to half of its volume with the formulation. In anotherembodiment, a formulation of the invention is a lyophilized formulationcomprising a protein of interest of the invention, wherein at leastabout 90%, at least about 95%, at least about 97%, at least about 98%,or at least about 99% of the protein of interest may be recovered from avial upon subjecting the formulation to three freeze/thaw cycles whereinthe vial is filled to half of its volume with said formulation. In afurther embodiment, a formulation of the invention is a lyophilizedformulation comprising a protein of interest of the invention, whereinat least about 90%, at least about 95%, at least about 97%, at leastabout 98%, or at least about 99% of the protein of interest may berecovered by reconstituting a lyophilized cake generated from saidformulation.

In one embodiment, a reconstituted liquid formulation may comprise aprotein of interest (e.g., antibody) at the same concentration as thepre-lyophilized liquid formulation.

In another embodiment, a reconstituted liquid formulation may comprise aprotein of interest (e.g., antibody) at a higher concentration than thepre-lyophilized liquid formulation, e.g., about 2 fold, about 3 fold,about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold,about 9 fold, or about 10 fold higher concentration of a protein ofinterest than the pre-lyophilized liquid formulation.

In yet another embodiment, a reconstituted liquid formulation maycomprise a protein of interest (e.g., antibody) of the invention at alower concentration than the pre-lyophilized liquid formulation, e.g.,about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold,about 7 fold, about 8 fold, about 9 fold or about 10 fold lowerconcentration of a protein of interest than the pre-lyophilized liquidformulation.

The pharmaceutical formulations of the low impurity compositions, forexample, low aggregate compositions, of the invention are typicallystable formulations, e.g., stable at room temperature.

The terms “stability” and “stable” as used herein in the context of aformulation comprising a protein of interest (e.g., an antibody) of theinvention refer to the resistance of the protein of interest in theformulation to aggregation, degradation or fragmentation under givenmanufacture, preparation, transportation and storage conditions. The“stable” formulations of the invention retain biological activity undergiven manufacture, preparation, transportation and storage conditions.The stability of the protein of interest can be assessed by degrees ofaggregation, degradation or fragmentation, as measured by HPSEC, staticlight scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR),circular dichroism (CD), urea unfolding techniques, intrinsic tryptophanfluorescence, differential scanning calorimetry, and/or ANS bindingtechniques, compared to a reference formulation. For example, areference formulation may be a reference standard frozen at −70° C.consisting of 10 mg/ml of a protein of interest of the invention in PBS.

Therapeutic formulations of the low impurity compositions, for example,low aggregate compositions, of the invention may be formulated for aparticular dosage. Dosage regimens may be adjusted to provide theoptimum desired response (e.g., a therapeutic response). For example, asingle bolus may be administered, several divided doses may beadministered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the protein of interest (e.g., antibody) and theparticular therapeutic effect to be achieved, and (b) the limitationsinherent in the art of compounding such a protein of interest for thetreatment of sensitivity in individuals.

Therapeutic compositions of the low impurity compositions, for example,low aggregate compositions, of the invention can be formulated forparticular routes of administration, such as oral, nasal, pulmonary,topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods knownin the art of pharmacy. The amount of active ingredient which can becombined with a carrier material to produce a single dosage form willvary depending upon the subject being treated, and the particular modeof administration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the composition which produces a therapeutic effect.By way of example, in certain embodiments, the proteins of interest(including fragments of the protein of interest) are formulated forintravenous administration. In certain other embodiments, the proteinsof interest (e.g., antibodies), including fragments of the proteins ofinterest (e.g., antibody fragments) are formulated for local delivery tothe cardiovascular system, for example, via catheter, stent, wire,intramyocardial delivery, intrapericardial delivery, or intraendocardialdelivery.

Formulations of the low impurity compositions, for example, lowaggregate compositions, of the invention which are suitable for topicalor transdermal administration include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Theactive compound may be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required (U.S. Pat. Nos. 7,378,110;7,258,873; 7,135,180; 7,923,029; and US Publication No. 20040042972).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the low impurity compositions, for example, lowaggregate compositions, of the invention may be varied so as to obtainan amount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

In certain embodiments, the proteins of interest (e.g., antibodies) ofthe invention can be formulated to ensure proper distribution in vivo.For example, the blood-brain barrier (BBB) excludes many highlyhydrophilic compounds. To ensure that the therapeutic compounds of theinvention can cross the BBB (if desired), they can be formulated, forexample, in liposomes. For methods of manufacturing liposomes, see,e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; 5,399,331. The liposomes maycomprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res.Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett.357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);surfactant Protein A receptor (Briscoe et al. (1995) Am. J. Physiol.1233:134), different species of which may comprise the formulations ofthe invention, as well as components of the invented molecules; p120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; inanother embodiment, the liposomes include a targeting moiety. In anotherembodiment, the therapeutic compounds in the liposomes are delivered bybolus injection to a site proximal to the desired area. Whenadministered in this manner, the composition must be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and may be preserved against thecontaminating action of microorganisms such as bacteria and fungi.Additionally or alternatively, the proteins of interest (e.g.,antibodies) of the invention may be delivered locally to the brain tomitigate the risk that the blood brain barrier slows effective delivery.

In certain embodiments, the low impurity compositions, for example, lowaggregate compositions, of the invention may be administered withmedical devices known in the art. For example, in certain embodiments aprotein of interest (e.g., antibody) or a fragment of protein ofinterest (e.g., antibody fragment) is administered locally via acatheter, stent, wire, or the like. For example, in one embodiment, atherapeutic composition of the invention can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;4,790,824; 4,596,556. Examples of well-known implants and modules usefulin the present invention include: U.S. Pat. No. 4,487,603, whichdiscloses an implantable micro-infusion pump for dispensing medicationat a controlled rate; U.S. Pat. No. 4,486,194, which discloses atherapeutic device for administering medicants through the skin; U.S.Pat. No. 4,447,233, which discloses a medication infusion pump fordelivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Many other such implants, delivery systems, andmodules are known to those skilled in the art.

The efficient dosages and the dosage regimens for the reduced level ofat least one impurity compositions of the invention depend on thedisease or condition to be treated and can be determined by the personsskilled in the art. One of ordinary skill in the art would be able todetermine such amounts based on such factors as the subject's size, theseverity of the subject's symptoms, and the particular composition orroute of administration selected.

Alternative Formulations Containing the Low Impurity Compositions of theInvention Alternative Aqueous Formulations

The invention also provides a low impurity composition, for example alow aggregate composition, formulated as an aqueous formulationcomprising a protein of interest and water, as described in U.S. Pat.No. 8,420,081, the contents of which are hereby incorporated byreference. In these aqueous formulations, the protein of interest isstable without the need for additional agents. This aqueous formulationhas a number of advantages over conventional formulations in the art,including stability of the protein of interest in water without therequirement for additional excipients, increased concentrations ofprotein of interest without the need for additional excipients tomaintain solubility of the protein of interest, and low osmolality.These also have advantageous storage properties, as the proteins ofinterest in the formulation remain stable during storage, e.g., storedas a liquid form for more than 3 months at 7° C. or freeze/thawconditions, even at high protein of interest concentrations and repeatedfreeze/thaw processing steps. In one embodiment, formulations describedherein include high concentrations of proteins of interest such that theaqueous formulation does not show significant opalescence, aggregation,or precipitation.

In one embodiment, an aqueous low impurity composition comprising aprotein of interest, e.g., an antibody, an anti-TNFα antibody or antigenbiding portion thereof, and water is provided, wherein the formulationhas certain characteristics, such as, but not limited to, lowconductivity, e.g., a conductivity of less than about 2.5 mS/cm, aprotein of interest concentration of at least about 10 μg/mL, anosmolality of no more than about 30 mOsmol/kg, and/or the protein ofinterest has a molecular weight (Mw) greater than about 47 kDa. In oneembodiment, the formulation has improved stability, such as, but notlimited to, stability in a liquid form for an extended time (e.g., atleast about 3 months or at least about 12 months) or stability throughat least one freeze/thaw cycle (if not more freeze/thaw cycles). In oneembodiment, the formulation is stable for at least about 3 months in aform selected from the group consisting of frozen, lyophilized, orspray-dried.

In one embodiment, the formulation has a low conductivity, including,for example, a conductivity of less than about 2.5 mS/cm, a conductivityof less than about 2 mS/cm, a conductivity of less than about 1.5 mS/cm,a conductivity of less than about 1 mS/cm, or a conductivity of lessthan about 0.5 mS/cm.

In another embodiment, low impurity compositions included in theformulation have a given concentration, including, for example, aconcentration of at least about 1 mg/mL, at least about 10 mg/mL, atleast about 50 mg/mL, at least about 100 mg/mL, at least about 150mg/mL, at least about 200 mg/mL, or greater than about 200 mg/mL. Inanother embodiment, the formulation of the invention has an osmolalityof no more than about 15 mOsmol/kg.

The aqueous formulations described herein do not rely on standardexcipients, e.g., a tonicity modifier, a stabilizing agent, asurfactant, an anti-oxidant, a cryoprotectant, a bulking agent, alyroprotectant, a basic component, and an acidic component. In otherembodiments of the invention, the formulation contains water, one ormore proteins of interest, and no ionic excipients (e.g., salts, freeamino acids).

In certain embodiments, the aqueous formulation as described hereincomprise a low impurity composition comprising a protein of interestconcentration of at least 50 mg/mL and water, wherein the formulationhas an osmolality of no more than 30 mOsmol/kg. Lower limits ofosmolality of the aqueous formulation are also encompassed by theinvention. In one embodiment the osmolality of the aqueous formulationis no more than 15 mOsmol/kg. The aqueous formulation of the inventionmay have an osmolality of less than 30 mOsmol/kg, and also have a highprotein of interest concentration, e.g., the concentration of theprotein of interest is at least 100 mg/mL, and may be as much as 200mg/mL or greater. Ranges intermediate to the above recitedconcentrations and osmolality units are also intended to be part of thisinvention. In addition, ranges of values using a combination of any ofthe above recited values as upper and/or lower limits are intended to beincluded.

The concentration of the aqueous formulation as described herein is notlimited by the protein of interest size and the formulation may includeany size range of proteins. Included within the scope of the inventionis an aqueous formulation comprising at least 40 mg/mL and as much as200 mg/mL or more of a protein of interest, for example, 40 mg/mL, 65mg/mL, 130 mg/mL, or 195 mg/ml, which may range in size from 5 kDa to150 kDa or more. In one embodiment, the protein of interest in theformulation of the invention is at least about 15 kD in size, at leastabout 20 kD in size; at least about 47 kD in size; at least about 60 kDin size; at least about 80 kD in size; at least about 100 kD in size; atleast about 120 kD in size; at least about 140 kD in size; at leastabout 160 kD in size; or greater than about 160 kD in size. Rangesintermediate to the above recited sizes are also intended to be part ofthis invention. In addition, ranges of values using a combination of anyof the above recited values as upper and/or lower limits are intended tobe included.

The aqueous formulation as described herein may be characterized by thehydrodynamic diameter (D_(h)) of the proteins of interest in solution.The hydrodynamic diameter of the protein of interest in solution may bemeasured using dynamic light scattering (DLS), which is an establishedanalytical method for determining the D_(h) of proteins. Typical valuesfor monoclonal antibodies, e.g., IgG, are about 10 nm. Low-ionicformulations may be characterized in that the D_(h) of the proteins ofinterest are notably lower than protein of interest formulationscomprising ionic excipients. It has been discovered that the D_(h)values of antibodies in aqueous formulations made using thedisfiltration/ultrafilteration (DF/UF) process, as described in U.S.Pat. No. 8,420,081, using pure water as an exchange medium, are notablylower than the D_(h) of antibodies in conventional formulationsindependent of protein concentration. In one embodiment, antibodies inthe aqueous formulation as described herein have a D_(h) of less than 4nm, or less than 3 nm.

In one embodiment, the D_(h) of the protein of interest in the aqueousformulation is smaller relative to the D_(h) of the same protein ofinterest in a buffered solution, irrespective of protein of interestconcentration. Thus, in certain embodiments, a protein of interest in anaqueous formulation made in accordance with the methods describedherein, will have a D_(h) which is at least 25% less than the D_(h) ofthe protein of interest in a buffered solution at the same givenconcentration. Examples of buffered solutions include, but are notlimited to phosphate buffered saline (PBS). In certain embodiments,proteins of interest in the aqueous formulation of the invention have aD_(h) that is at least 50% less than the D_(h) of the protein ofinterest in PBS in at the given concentration; at least 60% less thanthe D_(h) of the protein of interest in PBS at the given concentration;at least 70% less than the D_(h) of the protein of interest in PBS atthe given concentration; or more than 70% less than the D_(h) of theprotein of interest in PBS at the given concentration. Rangesintermediate to the above recited percentages are also intended to bepart of this invention, e.g., about 55%, 56%, 57%, 64%, 68%, and soforth. In addition, ranges of values using a combination of any of theabove recited values as upper and/or lower limits are intended to beincluded, e.g., about 50% to about 80%.

In one aspect, the aqueous formulation includes the protein of interestat a dosage of about 0.01 mg/kg-10 mg/kg. In another aspect, the dosagesof the protein of interest include approximately 1 mg/kg administeredevery other week, or approximately 0.3 mg/kg administered weekly. Askilled practitioner can ascertain the proper dosage and regime foradministering to a subject.

Alternative Solid Unit Formulations

The invention also provides a low impurity composition of the inventionformulated as a stable composition of a protein of interest, e.g., anantibody, or antigen binding portion thereof, and a stabilizer, referredto herein as solid units, as described in Attorney Docket No.117813-31001, the contents of which are hereby incorporated by referenceherein.

Specifically, it has been discovered that despite having a highproportion of sugar, the solid units comprising the low impuritycompositions of the invention maintain structural rigidity and resistchanges in shape and/or volume when stored under ambient conditions,e.g., room temperature and humidity, for extended periods of time (e.g.,the solid units comprising the low impurity compositions of theinvention do not require storage in a sealed container) and maintainlong-term physical and chemical stability of the protein of interestwithout significant degradation and/or aggregate formation. Moreover,despite having a high proportion of sugar, the solid units comprisingthe low impurity compositions of the invention remain free-flowing whenstored under ambient conditions, e.g., room temperature and humidity,for extended periods of time, and yet are easily dissolved in an aqueoussolvent, e.g., water (e.g., the solid units require minimal mixing whencontacted with a solvent for reconstitution). Furthermore, the solidunits comprising the low impurity compositions of the invention may beprepared directly in a device for patient use. These properties, whencompared to existing techniques which require a vial containing alyophilized protein of interest provided as a cake (which may notstabilize a protein of interest for extended periods of time), aseparate vial for a diluent, one or more sterile syringes, and severalmanipulation steps, thus provides alternative approaches forreconstitution since the solid units comprising the low impuritycompositions of the invention may be provided, e.g., in a dual chamberedcartridge, to make reconstitution invisible during patient delivery.Furthermore, the solid units comprising the low impurity compositions ofthe invention are versatile in that they can be readily and easilyadapted for numerous modes of administration, such as parenteral andoral administration.

As used herein, the term “solid unit,” refers to a composition which issuitable for pharmaceutical administration and comprises a protein ofinterest, e.g., an antibody or peptide, and a stabilizer, e.g., a sugar.The solid unit comprising the low impurity compositions of the inventionhas a structural rigidity and resistance to changes in shape and/orvolume. In one embodiment, the solid unit comprising the low impuritycompositions of the invention is obtained by freeze-drying apharmaceutical formulation of a therapeutic protein of interest. Thesolid unit comprising the low impurity compositions of the invention maybe any shape, e.g., geometric shape, including, but not limited to, asphere, a cube, a pyramid, a hemisphere, a cylinder, a teardrop, and soforth, including irregularly shaped units. In one embodiment, the solidunit has a volume ranging from about 1 □l to about 20 □l. In anotherembodiment, the solid unit is not obtained using spray dryingtechniques, e.g., the solid unit is not a powder or granule.

As used herein, the phrase “a plurality of solid units” refers to acollection or population of solid units comprising the low impuritycompositions of the invention, wherein the collection comprises two ormore solid units having a substantially uniform shape, e.g., sphere,and/or volume distribution. A substantially uniform size distribution isintended to mean that the individual shapes and/or volumes of the solidunits comprising the low impurity compositions of the invention aresubstantially similar and not greater than a 10% standard deviation involume. For example, a plurality of solid units which are spherical inshape would include a collection of solid units having no greater than10% standard deviation from an average volume of the spheres. In oneembodiment, the plurality of solid units is free-flowing.

Kits and Articles of Manufacture Comprising the Low ImpurityCompositions of the Invention

Also within the scope of the present invention are kits comprising thelow impurity compositions of the invention and instructions for use. Theterm “kit” as used herein refers to a packaged product comprisingcomponents with which to administer the protein of interest (e.g.,antibody, or antigen-binding portion thereof)), of the invention fortreatment of a disease or disorder. The kit may comprise a box orcontainer that holds the components of the kit. The box or container isaffixed with a label or a Food and Drug Administration approvedprotocol. The box or container holds components of the invention whichmay be contained within plastic, polyethylene, polypropylene, ethylene,or propylene vessels. The vessels can be capped-tubes or bottles. Thekit can also include instructions for administering a protein ofinterest (e.g., an antibody) of the invention.

The kit can further contain one more additional reagents, such as animmunosuppressive reagent, a cytotoxic agent or a radiotoxic agent orone or more additional proteins of interest of the invention (e.g., anantibody having a complementary activity which binds to an epitope inthe TNFα antigen distinct from a first anti-TNFα antibody). Kitstypically include a label indicating the intended use of the contents ofthe kit. The term label includes any writing, or recorded materialsupplied on or with the kit, or which otherwise accompanies the kit.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with a liquid formulation or lyophilizedformulation of a protein of interest (e.g., an antibody or antibodyfragment thereof) of the invention. In one embodiment, a containerfilled with a liquid formulation of the invention is a pre-filledsyringe. In a specific embodiment, the formulations of the invention areformulated in single dose vials as a sterile liquid. For example, theformulations may be supplied in 3 cc USP Type I borosilicate amber vials(West Pharmaceutical Services—Part No. 6800-0675) with a target volumeof 1.2 mL. Optionally associated with such container(s) can be a noticein the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

In one embodiment, a container filled with a liquid formulation of theinvention is a pre-filled syringe. Any pre-filled syringe known to oneof skill in the art may be used in combination with a liquid formulationof the invention. Pre-filled syringes that may be used are described in,for example, but not limited to, PCT Publications WO05032627,WO08094984, WO9945985, WO03077976, US Patents U.S. Pat. No. 6,792,743,U.S. Pat. No. 5,607,400, U.S. Pat. No. 5,893,842, U.S. Pat. No.7,081,107, U.S. Pat. No. 7,041,087, U.S. Pat. No. 5,989,227, U.S. Pat.No. 6,807,797, U.S. Pat. No. 6,142,976, U.S. Pat. No. 5,899,889, U.S.Pat. No. 7,699,811, U.S. Pat. No. 7,540,382, U.S. Pat. No. 7,998,120,U.S. Pat. No. 7,645,267, and US Patent Publication No. US20050075611.Pre-filled syringes may be made of various materials. In one embodimenta pre-filled syringe is a glass syringe. In another embodiment apre-filled syringe is a plastic syringe. One of skill in the artunderstands that the nature and/or quality of the materials used formanufacturing the syringe may influence the stability of a proteinformulation stored in the syringe. For example, it is understood thatsilicon based lubricants deposited on the inside surface of the syringechamber may affect particle formation in the protein formulation. In oneembodiment, a pre-filled syringe comprises a silicone based lubricant.In one embodiment, a pre-filled syringe comprises baked on silicone. Inanother embodiment, a pre-filled syringe is free from silicone basedlubricants. One of skill in the art also understands that small amountsof contaminating elements leaching into the formulation from the syringebarrel, syringe tip cap, plunger or stopper may also influence stabilityof the formulation. For example, it is understood that tungstenintroduced during the manufacturing process may adversely affectformulation stability. In one embodiment, a pre-filled syringe maycomprise tungsten at a level above 500 ppb. In another embodiment, apre-filled syringe is a low tungsten syringe. In another embodiment, apre-filled syringe may comprise tungsten at a level between about 500ppb and about 10 ppb, between about 400 ppb and about 10 ppb, betweenabout 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb,between about 100 ppb and about 10 ppb, between about 50 ppb and about10 ppb, between about 25 ppb and about 10 ppb.

In certain embodiments, kits comprising proteins of interest (e.g.,antibodies) of the invention are also provided that are useful forvarious purposes, e.g., research and diagnostic including forpurification or immunoprecipitation of protein of interest from cells,detection of the protein of interest in vitro or in vivo. For isolationand purification of a protein of interest, the kit may contain anantibody coupled to beads (e.g., sepharose beads). Kits may be providedwhich contain the antibodies for detection and quantitation of a proteinof interest in vitro, e.g., in an ELISA or a Western blot. As with thearticle of manufacture, the kit comprises a container and a label orpackage insert on or associated with the container. The container holdsa composition comprising at least one protein of interest (e.g.,antibody) of the invention. Additional containers may be included thatcontain, e.g., diluents and buffers, control proteins of interest (e.g.,antibodies). The label or package insert may provide a description ofthe composition as well as instructions for the intended in vitro ordiagnostic use.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial, pre-filled syringe or other container that ishermetically sealed. In one embodiment, the unit dosage form is providedas a sterile particulate free solution comprising a protein of interest(e.g., an antibody) that is suitable for parenteral administration. Inanother embodiment, the unit dosage form is provided as a sterilelyophilized powder comprising a protein of interest (e.g., an antibody)that is suitable for reconstitution.

In one embodiment, the unit dosage form is suitable for intravenous,intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus,the invention encompasses sterile solutions suitable for each deliveryroute. The invention further encompasses sterile lyophilized powdersthat are suitable for reconstitution.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question, as well as how and how frequently toadminister the pharmaceutical. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, pre-filled syringe, sprayer, insufflator, intravenous (i.v.)bag, envelope and the like; and at least one unit dosage form of apharmaceutical agent contained within said packaging material, whereinsaid pharmaceutical agent comprises a liquid formulation containing aprotein of interest (e.g., an antibody). The packaging material includesinstruction means which indicate how that said protein of interest(e.g., antibody) can be used to prevent, treat and/or manage one or moresymptoms associated with a disease or disorder.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references, including literature references, issued patents,and published patent applications, as cited throughout this applicationare hereby expressly incorporated herein by reference. It should furtherbe understood that the contents of all the figures and tables attachedhereto are expressly incorporated herein by reference. The entirecontents of the following applications are also expressly incorporatedherein by reference: U.S. Provisional Patent Application 61/893,123,entitled “STABLE SOLID PROTEIN COMPOSITIONS AND METHODS OF MAKING SAME”,Attorney Docket Number 117813-31001, filed on Oct. 18, 2013; U.S.Provisional Application Ser. No. 61/892,833, entitled “LOW ACIDICSPECIES COMPOSITIONS AND METHODS FOR PRODUCING THE SAME USINGDISPLACEMENT CHROMATOGRAPHY”, Attorney Docket Number 117813-73602, filedon Oct. 18, 2013; U.S. Provisional Patent Application 61/892,710,entitled “MUTATED ANTI-TNFa ANTIBODIES AND METHODS OF THEIR USE”,Attorney Docket Number 117813-73802, filed on Oct. 18, 2013; U.S.Provisional Patent Application 61/893,068, entitled “LOW ACIDIC SPECIESCOMPOSITIONS AND METHODS FOR PRODUCING THE SAME”, Attorney Docket Number117813-73901, filed on Oct. 18, 2013; and U.S. Provisional PatentApplication 61/893,088, entitled “MODULATED LYSINE VARIANT SPECIES ANDMETHODS FOR PRODUCING AND USING THE SAME”, Attorney Docket Number117813-74101, filed on Oct. 18, 2013.

EXAMPLES General Materials and Methods

Except where noted, the materials and methods described in connectionwith the instant example were also employed in Examples 1-3, below.

Chromatography Method

Pre-packed media columns were used in the following experiments, exceptwhere specified. The column was equilibrated in a buffer system withappropriate pH and conductivity. The column load was prepared fromProtein A affinity chromatography eluates or concentrated CEXchromatography elutes by buffer exchange (if the eluates were withdifferent buffer components from the mixed mode target buffer system) oraddition of the stock solutions and/or water to obtain the target pH andconductivity as specified (if the eluates were with the same buffercomponents as the mixed mode target buffer system). The prepared loadmaterial was filtered and loaded on the column according to the targetload amount (g protein/L media) as specified followed by washing withthe equilibration buffer or buffer similar to equilibration buffer withvolumes as specified. The column Flow Through/Wash were collected asfractions or as a pool. HIC column was cleaned with 20% IsopropylAlcohol solution. 1M NaOH solution was used for column cleaning.

Buffer Preparation Method

Buffers were prepared targeting a specific salt concentration in abuffered system, and titrating to a specific pH with the conjugate acidor base. For example, an 800 mM Ammonium Sulfate (AmSO₄) pH 7.0 solutionwas made by dissolving AmSO4 salt in a 20 mM Tris-Acetate bufferedsolution, titrating with acetate, and subsequently bringing up to volumewith water to achieve the desired AmSO₄ concentration. Load samples wereprepared targeting a specific salt concentration by addition ofconcentrated salt solution in a buffered system, and titrating to aspecific pH with the conjugate acid or base. For example, an 800 mMAmSO4 pH 7.0 load was made by mixing the load in a 1:1 ratio with a 1600mM AmSO4 pH 7.0 stock buffer in a 40 mM Tris-Acetate, and subsequentlytitrating with Tris or acetate to achieve a final pH 7.0.

Size Exclusion Chromatography

The molecular weight distribution of collected samples were quantifiedaccording to the following methods. Size exclusion chromatography (SEC)was performed using a TSK-gel G3000SWxL, 5 μm, 125 Å, 7.8×300 mm column(Tosoh Bioscience) on an HP Agilent HPLC system. Injections were madeunder isocratic elution conditions using a mobile phase of 200 mM sodiumsulfate, 100 mM sodium phosphate, pH 6.8, and detected with absorbanceat 214 nm. Quantification is based on the relative area of detectedpeaks.

Host Cell Protein (HCP) ELISA

HCP assay is based on process specific antigen based ELISA. Sampledilutions were applied to achieve readings within the calibration range.The limit of quantitation of the assay is 0.625 ng/mL.

UV Spectroscopy A₂₈₀

UV A280 was used to determine protein concentrations for the samplespost protein A elution. The assay was performed on an Agilent UVSpectrophotometer following the method. The protein concentration wasdetermined using Beer-Lambert's Law, A=εlc, where A is Absorbance, c isthe extinction coefficient, 1 is the path length, and c is theconcentration. The absorbance was taken at 280 nm, the path length was 1cm, and the extinction coefficients were 1.39 for Adalimumab, 1.38 formAb B, and 1.43 for mAb C.

Example 1: Determining Operating Conditions Appropriate for anMAB:Media:Buffer Combination

The demonstration of the current invention for a specific antibody &media is provided in this example, and consists of: 1) Choosing a saltconcentration that allows product and impurities to bind at a given pH;2) Loading a small amount of protein to the column and then performing alinear gradient elution by decreasing the salt concentration; 3)Determining salt concentration range in which the protein elutes fromthe HIC media.

In this example, adalimumab and GE CaptoPhenyl were chosen. The columnwas equilibrated at 1.1 M AmSO₄ pH 7.0 (Tris/Acetate) for 10 CVs.Adalimumab was prepared at 1.1 M AmSO₄ and loaded to the column at 20g-protein/L of resin. The column was washed with 10 CVs of theequilibration buffer. A linear gradient from 1.1M to 0M AmSO₄ pH 7.0(Tris/Acetate) over 20CVs was performed. The process chromatogram isshown in FIG. 4.

This process can be repeated for any given mAb-media combination for agiven buffer system. Table 1 shows the DOE parameters determined usingthe method described above for adlimumab in AmSO4 pH 7.0 (Tris/acetate)for 3 different HIC adsorbents.

TABLE 1 Example Experimental Design Scope determined from LGE withdifferent resins Adlimumab - Ammonium Sulfate pH 7.0 (Tris/Acetate)Resin Buffer Concentration Range Tosoh Hexyl 250-750 mM GE CaptoPhenyl300-650 mM GE Butyl FF 800-950 mM

In practicing the current invention, the aggregate reduction desired canbe achieved by appropriate pooling of the load and wash fractions. Bycollecting and subsequently determining the product quality of eachfraction throughout the load and wash, the accumulative aggregatereduction and accumulative yield can be calculated using the weightedaverages up to a given fraction. Additionally, the instantaneous yieldcan be estimated by comparing the protein recovered against the totalprotein loaded to the column at a given fraction. Sample calculationsare shown below:

Sample Calculation A: Accumulative Yield Up to a Given Fraction

${{Accumulative}\mspace{14mu} {Yield}} = \frac{{Accumulated}\mspace{14mu} {Protein}\mspace{14mu} {Mass}\mspace{14mu} {Recovered}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {Fraction}}{{Total}\mspace{14mu} {Mass}\mspace{14mu} {Protein}\mspace{14mu} {Load}}$

Sample Calculation B: Accumulative Aggregate Reduction Up to a GivenFraction

${{Accumulative}\mspace{14mu} {Aggregate}\mspace{14mu} {reduction}} = {{\ldots \mspace{14mu} {Load}\mspace{14mu} {Agg}\mspace{14mu} \%} - \frac{{Accumulated}\mspace{14mu} {Aggregate}\mspace{14mu} {Mass}\mspace{14mu} {Recovered}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {Fraction}}{{Accumulated}\mspace{14mu} {Total}\mspace{14mu} {Protein}\mspace{14mu} {Mass}\mspace{14mu} {Recovered}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {Fraction}}}$

Sample Calculation C: Instantaneous Yield Up to a Given Fraction

${{Instantaneous}\mspace{14mu} {Yield}} = \frac{{Accumulated}\mspace{14mu} {Protein}\mspace{14mu} {Mass}\mspace{14mu} {Recovered}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {Fraction}}{{Total}\mspace{14mu} {Protein}\mspace{14mu} {Mass}\mspace{14mu} {Loaded}\mspace{14mu} {to}\mspace{14mu} {Column}\mspace{14mu} {at}\mspace{14mu} {Fraction}}$

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   -   1. For a given salt concentration and optionally pH and HIC        media.    -   2. Loading the HIC media in excess of the dynamic binding        capacity for the product for the given condition.    -   3. Washing the column with a buffer containing a similar salt        concentration and optionally pH used for the equilibration and        loading steps.    -   4. Collecting fractions throughout the loading and wash steps        and subsequently determining the product quality profile (e.g.        Aggregate, HCP etc.)

In this example, adalimumab and GE CaptoPhenyl were chosen. Theexperiment was performed at 400 mM sodium citrate (NaCit) pH 5.6. Thecolumn was equilibrated with 400 mM NaCit pH 5.6 for 10 CVs. Adalimumabwas prepared at 400 mM NaCit pH 5.6 and loaded to the column at 500g-protein/L-resin. The column was washed with 7 CVs of the equilibrationbuffer. The process chromatogram is shown in FIG. 5. Fractions werecollected and analyzed for product quality and the accumulative yieldand accumulative aggregate reduction calculated, shown in Table 2. Fromthis example, it is clear to one skilled in the art to determine a runcondition which delivers a targeted product quality and/or step yield.

This general approach is used to evaluate the performance for a givenoperating condition for any resin/mAb/buffer combination.

TABLE 2 Accumulative Yield and Aggregate Reduction from FIG. 5 FractionLoad Accumulative Recovery Accumulative ΔAgg D1  8 g/L  0% 0.82% D2  45g/L  4% 0.77% D3  82 g/L 12% 0.71% D4 119 g/L 19% 0.67% D5 156 g/L 26%0.62% D6 193 g/L 33% 0.56% D7 231 g/L 41% 0.51% E1 268 g/L 48% 0.47% E2305 g/L 55% 0.43% E3 342 g/L 62% 0.40% E4 379 g/L 70% 0.37% E5 416 g/L77% 0.34% E6 454 g/L 84% 0.32% E7 491 g/L 91% 0.29% F1 500 g/L 93% 0.29%F2 WASH 99% 0.28% F3 WASH 100%  0.28% F4 WASH 101%  0.29% F5 WASH 101% 0.29%

Example 2: Demonstration of Aggregate Reduction with HIC Resins

This data set is compiled to demonstrate the aggregate reductionachieved with six different HIC adsorbents. Each resin was evaluatedwith a 500 g/L load of adalimumab at a NaCit concentration near, andslightly higher than, the peak elution concentration determined from theprocess outlined in Example 1. Table 3 outlines the results from theseexperiments.

TABLE 3 Effect of HIC Resins on Aggregate Reduction of Adalimumab HICResin NaCit, pH 5.6 ΔAgg Yield Butyl 400 mM 1.5% 99.8% 450 mM 1.2% 85.7%Hexyl 240 mM 1.2% 93.9% 300 mM 1.1% 100.9% Phenyl 400 mM 1.5% 96.5% 450mM 1.2% 90.7% Octyl 350 mM 0.4% 98.5% 400 mM 0.1% 103.3% GE Butyl FF 550mM 1.2% 88.1% 600 mM 1.7% 83.0% PPG 450 mM 0.2% 97.5% 600 mM 1.0% 38.1%

Example 3: Demonstration of Aggregate Reduction with Other Antibodies,MAB B and MAB C

Aggregate reduction technology of the current invention has beendemonstrated with multiple antibodies using HIC adsorbents. Antibodieshave different hydrophobic properties, leading to interaction behavioron a HIC column that differs from one antibody to another. Therefore theimpact of salt type and concentration is different for each antibody.

Table 4 and Table 5, presented below, provide the data obtained for mAbB and mAB C. The data clearly demonstrates that the aggregate reductiontechnology is effective for alternatives to adalimumab.

TABLE 4 Aggregate reduction for mAb B, pI ~9.1 HIC Resin AmSO4, pH 5.0ΔAgg Yield Hexyl 370 mM 0.8% 100%  710 mM 0.6% 93% Phenyl 340 mM 0.6%95% 790 mM 0.5% 95% Butyl 840 mM 0.6% 99% 1000 mM  0.6% 96%

TABLE 5 Aggregate reduction for mAb C, pI ~7.0 HIC Resin AmSO4, pH 5.0ΔAgg Yield Hexyl  80 mM 5.0% 89.0% 330 mM 4.5% 99.8% Phenyl 130 mM 3.5%92.8% 480 mM 2.9% 92.8% Butyl 690 mM 5.2% 93.5% 880 mM 5.4% 87.9%

Example 4: Demonstration of Aggregate Reduction with Different SaltConcentrations—Adalimumab

Ion concentration is a key variable in the performance of hydrophobicinteraction chromatography. For every combination of antibody/resin/pHthere is a range of ion concentrations that provide aggregate reduction;the strategy outlined in Example 1. can be followed to determine theaggregate reduction and the corresponding recovery for each saltconcentration.

Table 6, below, shows the effect of salt concentration on aggregatereduction and step yield. In this example CaptoPhenyl and adalimumabwere chosen, and evaluated at a loading of 200-500 g/L in NaCit pH 5.6at the concentration specified. The data demonstrates that the aggregatereduction can be effectively achieved over a range of saltconcentrations, and that the salt concentration and column loading canbe balanced to achieve a desired step yield and final product quality

TABLE 6 Effect of Ion Concentration on Aggregate Reduction NaCit pH 5.6Load Yield ΔAgg 300 mM 200 g/L 92% 0.59% 350 g/L 96% 0.33% 500 g/L 97%0.24% 400 mM 200 g/L 90% 0.76% 350 g/L 94% 0.43% 500 g/L 96% 0.35% 500mM 200 g/L 85% 1.09% 350 g/L 91% 0.97% 500 g/L 94% 0.86%

Example 5: Demonstration of Aggregate Reduction with Different BufferSystems with Adalimumab

In addition to the salt concentration, the salt anion and cation typesare key variables in hydrophobic interaction chromatography. Theinvention has been demonstrated with ammonium sulfate, sodium sulfate,and sodium citrate. As one skilled in the art would appreciate theoptimal salt concentration and optionally pH are different for each salttype and was derived by using the strategy outlined in Example 1. Table7 shows the data of aggregate reduction and corresponding recovery forthe different anion/cation types and different HIC adsorbents.

TABLE 7 Effect of Anion/Cation Type Aggregate Reduction Resin BufferSystem Load Yield ΔAgg CaptoPhenyl 630 mM AmSO4 pH 7.0 300 g/L 95% 2.1%300 mM AmSO4 pH 7.0 300 g/L 99% 1.1% 425 mM NaSO4 pH 7.0 300 g/L 95%1.9% 240 mM NaSO4 pH 7.0 300 g/L 101%  1.1% 500 mM NaCit pH 5.6 350 g/L91% 1.0% 300 mM NaCit pH 5.6 350 g/L 96% 0.2% Tosoh Hexyl 725 mM AmSO4pH 7.0 300 g/L 94% 1.7% 275 mM AmSO4 pH 7.0 300 g/L 103%  0.9% 460 mMNaSO4 pH 7.0 300 g/L 97% 0.7% 180 mM NaSO4 pH 7.0 300 g/L 101%  0.6% 440mM NaCit pH 5.6 300 g/L 87% 0.5% 150 mM NaCit pH 5.6 300 g/L 97% 0.5%Butyl FF 800 mM AmSO4 pH 7.0 300 g/L 100%  0.7% 1000 mM AmSO4 pH 7.0 300g/L 94% 1.6% 750 mM NaSO4 pH 7.0 300 g/L 96% 1.8% 700 mM NaSO4 pH 7.0300 g/L 101%  1.7% 700 mM NaCit pH 5.6 300 g/L 98% 1.6% 600 mM NaCit pH5.6 300 g/L 95% 1.5%

Example 6: Demonstration of Aggregate Reduction with Different Loading

Furthermore, the strategy outlined in Example 1. to reduce aggregatesthrough careful control of ion concentration, ion type, HIC adsorbent,and pH can be applied to various ranges of protein loading. Aggregatereduction for a range of protein loadings (e.g. 250-700 g/L) forCaptoPhenyl using a 400 mM NaCit pH 5.6 buffer is shown in Table 8,displaying a robust aggregate reduction across an expansive loadingrange.

TABLE 8 Impact of Column loading Load Yield ΔAgg ΔAgg/LoadAgg 250 g/L 95% 0.29% 87% 500 g/L 100% 0.25% 77% 700 g/L 100% 0.21% 65%

Example 7: Demonstration of Aggregate Reduction with Different LoadConcentration—Adalimumab

In addition to the strategy outlined in Example 6. to reduce aggregatesthrough careful control of ion concentration, ion type, and HICadsorbent, it has been shown that the concentration of the load proteincan have an effect on aggregate reduction. In this example, a feedstream was serial diluted to cover a range of load concentrations from 4to 15 mg/mL and loaded at 500 g/L to a CaptoPhenyl column in 400 mMNaCit pH 5.6. The effect of decreasing the concentration of the loadprotein is shown in FIG. 6.

Example 8: Demonstration of HCP Reduction in Addition to AggregateReduction

HIC chromatography can also be effective in reducing host cell protein(HCP) levels. In the present invention, it has been demonstrated thatHCP levels can be effectively reduced under operating conditionsselected for aggregate reduction.

Table 9 shows HCP removal achieved along with aggregate reduction. Thedata clearly shows that other process related substances/impurities canbe achieved using the current invention on the HIC adsorbents, and hencefunctions as an effective polishing step in the large scale purificationof monoclonal antibodies.

TABLE 9 HCP Removal during HIC Chromatography HCP NaCit pH 5.6 LoadYield ΔAgg Load Pool 300 mM 200 g/L 92% 0.59% 1398 ng/mg NA 350 g/L 96%0.33% 150 ng/mg 500 g/L 97% 0.24% 348 ng/mg 200 g/L 99% 0.34%  38 ng/mg 5 ng/mg 400 mM 200 g/L 90% 0.76% 1599 ng/mg 104 ng/mg 350 g/L 94% 0.43%148 ng/mg 500 g/L 96% 0.35% 350 ng/mg 350 g/L 97% 0.35%  38 ng/mg  6ng/mg 500 mM 200 g/L 85% 1.09% 1528 ng/mg 169 ng/mg 350 g/L 91% 0.97%203 ng/mg 500 g/L 94% 0.86% 301 ng/mg 500 g/L 87% 0.35%  38 ng/mg  11ng/mg

Example 9: Demonstration of Impact of Dynamic and Equilibrium Binding

In the HIC-based separation strategies described herein, the measureddynamic binding capacity (DBC), which is conventionally measured at 10%breakthrough, was found to be greater than the amount of protein thatremained bound after washing the column (a.k.a equilibrium bindingcapacity, EBC) with a buffer with similar pH and salt concentration tothe equilibration and load conditions. For example, but not by way oflimitation, FIG. 10 shows an example of the DBC and EBC for the datapresented in FIG. 5. In addition, Table 10 shows effect of salt type,concentration, and HIC resin on DBC and EBC values for Adalimumab.

TABLE 10 Comparison of DBC and EBC values for Adilmumab Resin BufferSystem ΔAgg DBC EBC CaptoPhenyl 630 mM AmSO4 pH 7.0 2.1% 27 g/L 16 g/L300 mM AmSO4 pH 7.0 1.1%  6 g/L  4 g/L 425 mM NaSO4 pH 7.0 1.9% 22 g/L15 g/L 240 mM NaSO4 pH 7.0 1.1%  6 g/L  4 g/L Butyl FF 1000 mM AmSO4 pH7.0 1.6% 17 g/L 11 g/L 800 mM AmSO4 pH 7.0 0.7%  4 g/L  4 g/L 750 mMNaSO4 pH 7.0 1.8% 29 g/L 13 g/L 700 mM NaSO4 pH 7.0 1.7% 22 g/L 11 g/L700 mM NaCit pH 5.6 1.6% 39 g/L 24 g/L 600 mM NaCit pH 5.6 1.5% 17 g/L11 g/L

Example HIC 10: Combinations of Hic with Alternative SeparationStrategies

The methods described herein for reducing aggregates using HIC can beused as an independent operation or in combination with other processsteps that provide additional aggregate reduction or those providingadditional complementary and supplementary purification. Data forspecific separation strategies is provided in Tables 11 and 12. Forexample, but not by way of limitation, the following processcombinations can be used:

1. Affinity→HIC

2. Affinity→AEX→HIC

3. Affinity→Mixed Mode→HIC

TABLE 11 Aggregate reduction with different source materials HCP LoadSource Buffer Condition Load Yield ΔAgg LRF Protein A 400 mM NaCit pH5.6 500 g/L 96% 1.49% NA Eluate 450 mM NaCit pH 5.6 500 g/L 91% 1.22% NAProteinA/ 300 mM NaCit pH 5.6 200 g/L 92% 0.59% 1.0 AEX FTW 400 mM NaCitpH 5.6 350 g/L 94% 0.43% 1.0 500 mM NaCit pH 5.6 500 g/L 94% 0.86% 0.7ProteinA/ 300 mM NaCit pH 5.6 200 g/L 99% 0.34% 0.8 Mixed Mode 400 mMNaCit pH 5.6 350 g/L 97% 0.35% 0.8 FTW 500 mM NaCit pH 5.6 500 g/L 87%0.35% 0.5

TABLE 12 Complete Process Train with Protein A Capture - AR, HMW and HCPreduction % HMW Process Yield (%) reduction HCP LRF Clarified Harvest97.00% n/a n/a Prt-A Eluate Pool 69.60% n/a 1.87 Viral Inactivated99.70% 0.07 0.39 Filtrate MM FT pool 91.90% 0.83 1.63 HIC FT-pool 98.50%0.23 0.46 VF(FT) Filtrate 96.10% No reduction 0.1 BDS (FT) 103.80% Noreduction 0.13

Example 11: Hybrid HIC Binding Mechanism

By estimating the partitioning coefficient K_(p), it can be demonstratedthat certain strategies described in the instant application do not fallunder the category of “Weak-Partitioning (WP)” or “Flow-Through Overload(FT)” modes as those are described in the art, e.g., US2007/0060741. Forexample, FIGS. 13A-13B depict the results of experiments whereinaliquots of resin are incubated with a load covering a range of proteinconcentrations at room temperature for 3 hours, after which the proteinsolution is then removed, and replaced with equilibration buffer (Washsimulation) and incubated at room temperature for 3 hours (repeated,Wash II). After each incubation, the concentration of the proteinsolution is measured and used to calculated the amount of protein ((A)monomer D2E7, a.k.a. Adalimumab, and (B) aggregate D2E7) bound to theresin (g protein/L resin) and plotted against the concentration of theprotein solution at the end of the incubation (e.g. equilibrium). FIGS.14A-14B depict the results outlined in FIGS. 13A-13B, highlighting thefact that at initial equilibrium a significant amount ofmonomer/aggregate is bound to the resin. However, after the proteinsolution is replaced with equilibration buffer (see arrow), the monomerde-sorbs from the resin and back into solution, whereas the aggregateremains bound.

FIGS. 15A-15B depict a determination of the binding monomer andaggregate D2E7 (based on data provided in FIGS. 13A-13B) by fitting theexperimental equilibrium binding data to the Langmuir Isotherm using theequation: q=(q_(max)×C_(equil)) (K_(a)+C_(equil)); where q=amount ofprotein bound to resin [=] g/L-resin; q_(max)=maximum amount of proteinbound to resin [=] g/L-resin; C_(equil)=solution concentration ofprotein [=] g/L-soln; and K_(d)=equilibrium dissociation constant.

By fitting the experimental data, the q_(max) and K_(d) for the monomerand the aggregates can be calculated.

$\begin{matrix}\underset{\_}{Species} & \underset{\_}{Q_{\max}\left\lbrack {{mg}\text{/}{mL}} \right\rbrack} & \underset{\_}{K_{d}\left\lbrack {{mg}\text{/}{mL}} \right\rbrack} & \underset{\_}{\;} \\{Monomer} & 41.9 & 0.47 & \; \\{Aggregate} & {\mspace{14mu} 6.0} & 0.01 & \;\end{matrix}\;$

Significantly, q_(max) for both monomer/aggregate and the K_(d) values(i.e. strength of binding) are similar to those of strong hydrophobicinteractions, therefore it is not expected for this interaction to be“reversible.” In addition, by calculating K_(p) where:

$\begin{matrix}\underset{\_}{Species} & \underset{\_}{Q_{\max}\left\lbrack {{mg}\text{/}{mL}} \right\rbrack} & \underset{\_}{K_{d}\left\lbrack {{mg}\text{/}{mL}} \right\rbrack} & \underset{\_}{K_{p} \equiv \frac{Q}{C} \cong \frac{Q_{\max}}{K_{d}}} \\{Monomer} & 41.9 & 0.47 & {\mspace{14mu} 90} \\{Aggregate} & {\mspace{14mu} 6.0} & 0.01 & 600\end{matrix}$

it is apparent that the instant technique does not fall within thecategory of flow-through (where K_(p)≤1) or weak portioning (whereK_(p)=1-10), but rather fall within the category of bind-elute (whereK_(p)≥10).

Example 12: Determination of Binding Capacity at Saturation

The following protocol exemplifies the determination of (i) apparentbinding capacity, i.e., the binding capacity at saturation (when outletprotein concentration equals inlet protein concentration) under flowconditions and (ii) the actual binding capacity, i.e., the amount ofprotein that remains bound after an isocratic wash.

A column packed with resin containing a hydrophobic interaction ligandwas equilibrated with a buffer at a given salt concentration and pH. Aprotein load in the same buffer condition as the equilibration solutionwas loaded to the column until the protein breaks through the column,and the protein concentration at the effluent of the column was equal tothe protein concentration at the inlet of the column (i.e., saturated).The column was then washed with the equilibration solution until theprotein concentration at the effluent was effectively zero. Theremaining protein bound to the column was then eluted with a buffercondition that will cause the protein to desorb from the resin.

Taking into account the void volume of the column and chromatographysystem, one can calculate the amount of protein bound to the column atthe saturation point by integrating the area above the breakthroughcurve at the effluent of the column (FIGS. 12A-12B). After the isocraticwash, one can calculate the protein that remained bound to the resin byintegrating the area under the curve of the elution peak.

The differences between these two values is the ‘reversible’ bindingcapacity, which is significant when compared to the binding capacityobserved at the saturation point (e.g., “apparent binding capacity”).This difference is also a function of the salt concentration, which isshown in FIG. 16. FIG. 16 is a comparison of apparent and actual boundprotein under flow conditions. Binding of the antibody during loading issignificant (>10 g/l). The majority (>65%) of the antibody monomer boundduring load desorbs during the isocratic wash (i.e., reversibly bound).The mass balance of the impurity demonstrates irreversible binding.

Example 13: Determination of Binding Capacity at Saturation

A column was conditioned and loaded, as described in Example 12, atdifferent inlet protein concentrations. In these experiments, theflow-through fractionated to determine the product quality at differenttimes during the loading and breakthrough. Using the protein mass andproduct quality for each of the fractions, the accumulative impurity(e.g., aggregate) breakthrough can be calculated using the weightedaverage:

${{Accumulative}\mspace{14mu} \% \mspace{14mu} {Aggregates}} = \frac{\left( {{Sum}\mspace{14mu} {of}\mspace{14mu} {total}\mspace{14mu} {aggregate}\mspace{14mu} {mass}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {fraction}_{i}} \right)}{\left( {{Sum}\mspace{14mu} {of}\mspace{14mu} {total}\mspace{14mu} {protein}\mspace{14mu} {mass}\mspace{14mu} {up}\mspace{14mu} {to}\mspace{14mu} {fraction}_{i}} \right)}$

This calculation can be plotted for each successive fraction (FIG. 8A)and used to compare different loading conditions. The EquilibriumBinding Isotherms for both the monomer and aggregate show that for allof the loading conditions (FIGS. 8B-8C), the monomer was in thenon-linear part of its binding isotherm (e.g., equilibrium bindingcapacity is independent of monomer concentration), and the aggregate wasin or near the linear part of its binding isotherm (e.g., equilibriumbinding capacity is dependent on aggregate concentration). The clearanceof the aggregate improves by decreasing the overall load proteinconcentration, even though this results in the resin having a lowerbinding capacity for the aggregate.

In FIG. 7, material from a single source was serially diluted to threedifferent protein concentrations and subsequently loaded to a column to500 g/L. This data clearly demonstrates that diluting the load materialresulted in a better aggregate clearance, even though the same amount ofimpurity was loaded in each case. This is non-intuitive, especially whenconsidering that diluting the load protein concentration results in alower overall binding capacity for the impurity as the impurity is inthe linear range of the equilibrium binding isotherm and therefore thebinding capacity decreases linearly with concentration.

FIG. 9 is a re-plot of the same data as in FIG. 7 to demonstrate that,for a given target impurity clearance, the recovery-yield can bemodulated by diluting the load material to a specific range.

1. A method for producing a preparation comprising a protein of interestand having a reduced level of at least one impurity, said methodcomprising: (a) contacting a sample comprising the protein of interestand at least one impurity, to a hydrophobic interaction chromatography(HIC) media, in the presence of a load buffer such that (i) a portion ofthe protein of interest binds to the HIC media and (ii) a substantialportion of the at least one impurity binds to the HIC media; (b)collecting a flow through fraction comprising the protein of interestunbound to the HIC media; (c) washing the HIC media with a wash bufferthat is substantially the same as the load buffer such that asubstantial portion of the protein of interest bound to the HIC media isreleased from the media; and (d) collecting a wash fraction comprisingthe protein of interest released from the HIC media, wherein each of theflow through and wash fractions comprise the protein of interest andhave a reduced level of the at least one impurity.
 2. The method ofclaim 1, wherein the portion of the protein of interest binds to the HICmedia at (a) a Kp of greater than 10, 15, 20, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250 or 300; or(b) at a Kp of greater than
 20. 3. The method of claim 1, wherein theprotein of interest is adalimumab.
 4. The method of claim 1, wherein asubstantial portion of the impurity bound to the HIC media remains boundupon washing with the wash buffer.
 5. The method of claim 1, wherein theflow through and/or wash fractions are substantially free of the atleast one impurity.
 6. The method of claim 1, wherein the at least oneimpurity is an aggregate of the protein of interest, optionally, amultimer, a dimer, a trimer, a tetramer, an oligomer or other highmolecular weight species.
 7. The method of claim 1, wherein the proteinof interest is adalimumab and the at least one impurity is an aggregateof adalimumab, optionally, selected from the group consisting ofmultimer 1, multimer 2 and multimer
 3. 8. The method of claim 1, whereinthe impurity is a process-related impurity or a product-relatedsubstance.
 9. The method of claim 8, wherein the process-relatedimpurity is selected from the group consisting of a host cell protein, ahost cell nucleic acid, a media component, and a chromatographicmaterial.
 10. The method of claim 8, wherein the product-relatedsubstance is selected from the group consisting of a charge variant, anacidic variant, a basic variant, a lysine variant species, an aggregateof the protein of interest, a fragment of the protein of interest, an Fcfragment of the protein of interest, a Fab fragment of the protein ofinterest, a modified protein, a deamidated protein, and a glycosylatedprotein.
 11. The method of claim 1, wherein the impurity is an acidicspecies (AR), optionally, selected from the group consisting of AR1,AR2, a charge variant, a structure variant, a fragmentation variant, aprocess-related impurity and a product-related impurity.
 12. The methodof claim 1, wherein the protein of interest is an antibody orantigen-binding fragment thereof, a soluble protein, a membrane protein,a structural protein, a ribosomal protein, an enzyme, a zymogen, anantibody molecule, a humanized antibody or antigen-binding portionthereof, a human antibody or antigen-binding portion thereof, a chimericantibody or antigen-binding portion thereof, a multivalent antibody, acell surface receptor protein, a transcription regulatory protein, atranslation regulatory protein, a chromatin protein, a hormone, a cellcycle regulatory protein, a G protein, a neuroactive peptide, animmunoregulatory protein, a blood component protein, an ion gateprotein, a heat shock protein, an antibiotic resistance protein, afunctional fragment of any of the preceding proteins, anepitope-containing fragment of any of the preceding proteins, andcombinations thereof.
 13. The method of claim 1, further comprisingrepeating steps (a)-(d) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 or 20 times using the flow through fraction, wash fraction,or combination thereof having a reduced level of the at least oneimpurity
 14. The method of claim 1, wherein the flow through fractionand the wash fraction are combined.
 15. The method of claim 1, wherein(a) the portion of the protein of interest that binds to the HIC mediais at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80% orat least about 90% of the protein of interest in the sample; (b) thesubstantial portion of the protein of interest released from the HICmedia upon washing with the wash buffer is at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90% or about 100%of the amount of protein of interest bound to the HIC media; (c) theaccumulative yield of the protein of interest in the flow throughfraction and/or wash fraction is at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100%; (d) the accumulative yield of the protein ofinterest in any one flow through fraction and/or wash fraction is atleast about 4%, at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85, at least about 90%, at leastabout 95% or about 100%; (e) the substantial portion of the at least oneimpurity that binds to the HIC media is at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95% or about 100% of the at least one impurityin the sample; (f) the reduced level of the at least one impurity of theflow through fraction and/or wash fraction is at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95% or about 100% of the at least oneimpurity in the sample; (g) the accumulative aggregate reduction of theat least one impurity in any one flow through fraction and/or washfraction is at least about 0.1%, at least about 0.2%, at least about0.5%, at least about 1.0%, at least about 2.0%, at least about 3.0%, atleast about 4.0%, at least about 5.0%, at least about 10.0%, or at leastabout 20.0%; (h) the accumulative aggregate reduction of the at leastone impurity in the flow through fraction and/or wash fraction is atleast about 0.1%, at least about 0.2%, at least about 0.5%, at leastabout 1.0%, at least about 2.0%, at least about 3.0%, at least about4.0%, at least about 5.0%, at least about 10.0%, or at least about20.0%; and/or (i) the level of the at least one impurity is reduced byat least 60%, at least 70%, at least 80%, at least 90%, or at least 95%of the at least one impurity in the sample.
 16. The method of claim 1,wherein (a) the at least one impurity binds to the HIC media at a Kp ofgreater than 250, greater than 300, greater than 400, greater than 500,greater than 600, greater than 700, greater than 800, greater than 900,or greater than 1000; (b) the protein of interest and the at least oneimpurity have a Kp ratio less than 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4,1:3 or 1:2; (c) the Kd for the binding of the protein of interest to theHIC media is at least about 0.2, at least about 0.3, at least about 0.4,at least about 0.5, or at least about 0.6; (d) the Kd for the binding ofthe at least one impurity to the HIC media is less than or equal toabout 0.001, about 0.005, about 0.01, about 0.02, about 0.05, about 0.1,about 0.15 or about 0.2; (e) the Kd for the binding of the protein ofinterest to the HIC media is less than 50, 45, 40, 35, 30, 25, 20, 15,10 or 5 times the Kd for the binding of the at least one impurity to theHIC media; (f) the protein of interest has a Qmax of at least about 20,at least about 30, at least about 40, at least about 50, at least about60 or at least about 100; and/or (g) the at least one impurity has aQmax of at least about 2, at least about 5, at least about 10, at leastabout 20, at least about 30 or at least about
 40. 17. The method ofclaim 1, wherein the HIC media either (i) comprises at least onehydrophobic ligand, optionally selected from the group consisting ofalkyl-, aryl-ligands, and butyl, hexyl, phenyl, octyl, or polypropyleneglycol ligands; and/or (ii) is selected from the group consisting ofCaptoPhenyl, Phenyl Sepharose™ 6 Fast Flow with low or highsubstitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™ HighPerformance, Fractogel™ EMD Propyl, Fractogel™ EMD Phenyl, Macro-Prep™Methyl, Macro-Prep™ t-Butyl, WP HI-Propyl (C3)™, Toyopearl™ ether,Toyopearl™ phenyl, Toyopearl™ butyl, ToyoScreen PPG, ToyoScreen Phenyl,ToyoScreen Butyl, ToyoScreen Hexyl, HiScreen Butyl FF, HiScreen OctylFF, and Tosoh Hexyl.
 18. The method of claim 1, wherein (a) the loadbuffer and/or wash buffer comprise a salt selected from the groupconsisting of a sulfate salt, a citrate salt, ammonium sulfate, sodiumsulfate, sodium chloride, ammonium chloride, sodium bromide and acombination thereof; (b) the load buffer and/or wash buffer comprise acation selected from the group consisting of Ba2+, Ca2+, Mg2+, Li+, Cs+,Na+, K+, Rb+, NH4+ and a combination thereof; (c) the load buffer and/orwash buffer comprise an anion selected from the group consisting ofPO43−, SO42−, CH3CO3−, Cl−, Br−, NO3−, ClO4−, I−, SCN− and a combinationthereof; (d) the load buffer and/or wash buffer comprise a salt having aconcentration of between about 50 mM and 2000 mM; (e) the load bufferand/or wash buffer have a pH between about 4.0 and 8.5 or between about5.0 and 7.0; (f) the load buffer and/or wash buffer have a pH of about4.0, about 4.5, about 5.0, about 5.5, about 6, about 6.5, about 7.0,about 7.5, about 8.0, or about 8.5; (g) the load buffer and/or washbuffer are the same; (h) the load buffer and/or wash buffer aresubstantially the same; and/or (i) the salt concentration and/or the pHof the wash buffer are within about 20%, 15%, 10% or 5% of the saltconcentration and/or pH of the loading buffer.
 19. The method of claim1, wherein (a) about 100 g to about 800 g of the sample are contactedper one liter of HIC media; (b) about 0.2 g to about 120 g of the atleast one impurity is contacted per one liter of HIC media; (c) thesample has a protein concentration of about 2 mg/ml to about 50 mg/ml;(d) the sample has a protein of interest concentration of about 2 mg/mlto about 50 mg/ml; and/or (e) the concentration of the at least oneimpurity in the sample is about 0.01 to about 5.0 mg/ml.
 20. The methodof claim 1, (a) wherein a precursor sample comprising the protein ofinterest has been subjected to affinity chromatography to generate thesample; and/or (b) further comprising subjecting the preparationcomprising a protein of interest and having a reduced level of oneimpurity to affinity chromatography, optionally wherein affinitychromatography is performed using affinity chromatographic mediaselected from the group consisting of Protein A, G, A/G, L media, andMabSuRe Protein A media.
 21. The method of claim 1, (a) wherein aprecursor sample comprising the protein of interest has been subjectedto ion exchange chromatography to generate the sample; and/or (b)further comprising subjecting the preparation comprising a protein ofinterest and having a reduced level of one impurity to ion exchangechromatography, optionally wherein ion exchange chromatography isperformed using ion exchange chromatography media selected from thegroup consisting of a cation exchange media and an anion exchange media.22. The method of claim 1, (a) wherein a precursor sample comprising theprotein of interest has been subjected to mixed mode chromatography togenerate the sample; and/or (b) further comprising subjecting thepreparation comprising a protein of interest and having a reduced levelof one impurity to mixed mode chromatography; optionally wherein themixed mode chromatography is performed using CaptoAdhere resin.
 23. Themethod of claim 1, (a) wherein a precursor sample comprising the proteinof interest has been subjected to a filtration step to generate thesample; and/or (b) further comprising subjecting the preparationcomprising a protein of interest and having a reduced level of oneimpurity to a filtration step; optionally, wherein the filtration stepis selected from the group consisting of a depth filtration step, ananofiltration step, an ultrafiltration step, and an absolute filtrationstep, or a combination thereof.
 24. The method of claim 1, wherein theHIC media has a dynamic binding capacity of at least about 2 g, at leastabout 5 g, at least about 10 g, at least about 20 g, at least about 30g, at least about 40 g, at least about 50 g, at least about 60 g, atleast about 70 g, at least about 90 g, or at least about 100 g of sampleper one liter of media.
 25. A method for producing a preparationcomprising adalimumab and having a reduced level of at least oneaggregate, said method comprising: (a) contacting a sample comprisingadalimumab and at least one aggregate, to a HIC media, in the presenceof a load buffer such that (i) a portion of the adalimumab in the samplebinds to the HIC media and (ii) a substantial portion of the at leastone aggregate binds to the HIC media; (b) collecting a flow throughfraction comprising the adalimumab unbound to the HIC media; (c) washingthe HIC media with a wash buffer that is substantially the same as theload buffer such that a substantial portion of the adalimumab bound tothe HIC media is released from the media; and (d) collecting a washfraction comprising the adalimumab released from the HIC media, whereineach of the flow through and wash fractions comprise adalimumab and havea reduced level of the at least one aggregate.
 26. The method of claim25, wherein adalimumab binds to the HIC media (a) at a Kp of greaterthan 10, 15, 20, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 220, 250 or 300; or (b) at a Kp of greater than 20.27. The method of claim 25, wherein the aggregate is multimer 1,multimer 2 or multimer
 3. 28. (canceled)
 29. A pharmaceuticalcomposition comprising a low-aggregate composition of adalimumab and apharmaceutically acceptable carrier.
 30. The pharmaceutical compositionof claim 29, wherein the composition comprises less than 5%, 4%, 3%,2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%,0.5%, 0.1% of aggregates.